CN101065136A - Prion protein binding materials and methods of use - Google Patents

Abstract

Prion protein binding materials and methods for using the binding materials to detect or remove a prion protein from a sample, such as a biological fluid or an environmental sample are disclosed. The binding materials are capable of binding to one or more forms of prion protein including cellular prion protein (PrPc), infectious prion protein (PrPsc), recombinant prion protein (PrPr), and proteinase resistant prion protein (PrPres). Prions from various species, including humans and hamsters, are bound by the binding materials.

Description

Prion protein binding materials and using method thereof

Technical field

The present invention relates to protein binding (binding) field, more specifically, relate to the method that detects or remove the Protein virus in the biological sample with bonded material of prion protein and application prion protein binding materials.

Background technology

Natural or cell prion protein " PrPc " is distributed widely in the mammal, and has special very conservative aminoacid sequence and protein structure.Think that infectious prion is to be made of the modified forms of normal cell (PrPc) prion protein, and be called " PrPsc ".Protein virus and other infectious agent have some common characteristics, but seem wherein not contain nucleic acid.On the contrary, think that the conformation change after the translation has caused noninfectious PrPc to change communicable PrPsc into, alpha-helix has wherein taken place be transformed into beta sheet.PrPc contains three α-Luo Xuanjiegous and beta sheet structure seldom; In contrast, PrPsc is rich in beta sheet.Believe that PrPc has caused the development of propagability spongiform encephalopathy (TSEs) to the transformation of PrPsc, wherein, PrPsc accumulates in central nervous system and simultaneous neuro pathology's variation and neurological dysfunction.PrPsc typically refers to " itching disease " form of prion protein, and thinks that this propagation and pathogenesis for propagable neurodegenerative disease in the animal and human is necessary, and is likely sufficient.

The object lesson of TSEs comprises itching disease, this affect sheep and goat; Mad cow disease (BSE); Propagable mink encephalopathy, cat spongiform encephalopathy and chronic consumption disease (CWD).In the people, the TSE disease can show as kuru, Ke Ya (family name) sick (or cortex-Basal ganglia-spinal cord degeneration syndrome (CJD)), Ge-Shi-Sha syndrome (Gerstmann-Stra ü ssler-ScheinKer (GSS)), the variant (vCJD) of mortality insomnia and CJD disease.In the mankind, occur vCJD recently, cause BSE very popular, and it most possibly is to cause owing to having eaten the food that derives from the livestock that has infected BSE or " bovine spongiform encephalopathy " in Britain.In Britain, 20th century the mid-80 early stage to the nineties because it is not clear to have absorbed the number of food of the domestic animal nervous tissue latent infection that has infected BSE.Because in the mankind, may surpass 20 years the incubation period of oral cavity infection disease, the actual incidence rate of vCJD may all can not manifest in a lot of years.So far, knownly surpass 150 people and infected this kind disease, mainly in Britain; Yet in Canada, France, Hong Kong, Israel, the Italy and the U.S. also reported this kind case.Britain worldwide exports infected cattle feed product and shows that all there is BSE in the whole world probably, and has brought the probability of vCJD therefrom.Corresponding to these observed results is to detect BSE in most of European countries, Japan, Canada, the U.S. and Israel.So, from the various different materials that comprise food, detect and remove communicable prion protein and just become extremely important.

The feature of all TSEs is: for such material, shortage can detected host immune response.So, also do not have the antibody special of determining up till now to TSCs.In addition, lack known nucleotide sequence and got rid of the diagnostic method of application polymerase chain reaction for the basis.So just do not have conventional serology to detect and to be used for determining infected animal.Recently, the technology based on immunology of improvement has been used for determining whether the brain at slaughtered animals has PrPsc.

Except picked-up derived from the infected product of cattle, blood transfusion and organ transplantation were the another kind of modes of propagating vCJD between the people.The risk of propagating vCJD by blood transfusion in the people is unknown at present, but, data based on experimental animal model, comprise with laboratory facilities made oral cavity infection sheep behind the BSE and the sheep that has infected itching disease natively and propagating, they have very possible probability as a result, and most probable is explained the propagation of interpersonal vCJD.Therefore do not resemble other people TSEs, PrPsc is present in vCJD patient's the lymphatic reticular endothelial cell system, has increased infectious substance and has been present in probability in the blood, and can propagate by blood transfusion.Other relates to passes through to transfuse blood that to improve the factor of propagating risk not clear, and it is contemplated that can be very high, much is exposed to the crowd of BSE, lacks for the preclinical diagnostic test of vCJD.In addition, as if vCJD toxicity adaptability along with the race in primates and Mus increases to some extent, this means that interpersonal propagation is more effective to people propagation than cattle.Therefore, needing certain methods to stop vCJD to pass through blood transfusion urgently propagates.Such measure can comprise the delaying of TSE material in the food of the early stage affirmation of infected donor and animal origin or the health product, removal and deactivation, the food of described animal origin or health product are applied to animal or people's current consumption or application, the blood source goods of people and Niu, organ transplantation.Very unfortunate, the infectiousness of TSE has significant resistance for the chemistry and the physical method of deactivation, and is difficult to find the selective inactivation method.

Determined much to be incorporated into the material of prion protein.Discovery examination from the combined peptide storehouse is incorporated into the part of octapeptide repetitive sequence (PHGGGWGQ) (SEQ ID NO:1), be present in all known mammal prion proteins, and found a series of parts, as described in PCT/US01/11150.Other material comprises and amyloid plaque such as the interactional part (Ingrosso of Congo red (Congo Red) amyloid plaque, L., Deng the people, Congo Red Prolongs the Incubation Period in Scrapie-infectedHamsters.J.Virology 69:506-508 (1995)); 4-iodine, 4-deoxidation amycin (Tagliavini, F. wait the people, Effectiveness of Anthracycline Against Experimental PrionDiseases in Syrian Hamsters.Science 276:1119-1122 (1997)); Amphotericin B, porphyrin and phthalocyanine (Priola, S.A. wait the people, Porphyrin and PhthalocyanineAntiscrapie Compounds, Science 287:1503-1506 (2000)); Metal (people such as Stockel, Biochemistry, 37,7185-7193 (1998)); Form the peptide of complex (referring to United States Patent (USP) 5 with the Prp interaction, 750,361, people and Soto such as Prusiner, C. wait the people, Reversionof Prion Protein Conformational Changes in Synthetic β-sheet BreakerPeptides, Lancet, 355:192-197 (2000)); Heparin and other poly-sulphation polyanion (Caughey, B., Deng the people, Binding of the Protease-sensitive Form of PrionProtein Prp to Sulphated Glycosaminoglycan and Congo Red, J.Virology68:2135-2141 (1994)); Antibody (Kascsak, R.J. wait the people, Immunodiagnosis ofPrion Disease, Immunological Invest.26:259-268 (1997)); With other albumen, for example plasminogen (Fischer, people such as M.B.., Binding of Disease-associatedPrion Protein to Plasminogen., Nature 408:479-483 (2000)).Ion-exchange chromatography has been used to the blood constitutent that the purification prion protein pollutes, for example hematochrome (U.S. Patent number 5,808,011, people such as Gawryl).Yet the chromatographic material of people such as Gawryl instruction is in conjunction with hematochrome, and collects the hematochrome of purification subsequently with gradient elution.At present, also not having material to be characterized fully or found can be in conjunction with the prion protein from multiple medium.

Up to the present, people TSE disease is 100% fatal.Unfortunately, be reported to therapeutic agent likely although comprise amphotericin, sulphation polyanion, Congo red dyestuff and the antibiotic a large amount of chemical compounds of anthracene nucleus class (anthracycline), have only appropriate potentiality but all these chemical compounds are verified for stoping prion protein to be bred, and the prion protein that does not have a kind of compound exhibits to go out for removal is pre-existing in from infected host in controlled clinical research there is any effect.Therefore, still press for novel treatment reagent at present.

The process that assembling from normal soluble protein to conformational change, soluble form and going is assembled is considered to the cause of disease of a variety of other diseases, and these diseases much are the diseases that belongs to the nervous system aspect.Change relation between the conformational change albumen into for the morbidity of disease and normal protein, also understand seldom.Except prion protein, the object lesson of these insoluble proteins also comprises: the β-amylaceous peptide among the amyloid plaque in Alzheimer (family name) disease and cerebral amyloid angiopathy; α in parkinson disease in the centration multilamellar corps ronds (Lu Yiti)-synuclein deposit, the Protein tau in the frontotemporal dementia in the neurofibrillary tangles, and Pick disease; Super (mistake) oxide dismutase in amyotrophic lateral sclerosis (spinal cord) lateral sclerosis; And the choreic huntingtin albumen of Heng Yandun (family name).These height insoluble proteins form usually by branchiess fibrillated aggregation, and its common feature is the βZhe Die conformation.

Need be convenient to distinguish or differentiate the albumen of two kinds or more kinds of not isomorphic map forms,, thereby understand this transforming process, and interactional specifically structure takes place in discovery and disease association form as the methodology of PrPc and PrPsc.The present methodology of distinguishing or differentiate proteic isoform comprises: under the chaotropic agent condition that exists as carbamide, and the different transfer rate on polyacrylamide gel, promptly horizontal carbamide gradient (TUG) gel; For the difference sensitivity of Protease Treatment such as E.C. 3.4.21.64 (PK), detect the PK-resistance digestion product of the PrPsc that is known as PrPres; Differential temperatures stability; Relative dissolubility in the nonionic detergent; And fibre structure is in conjunction with some chemical substance, as the ability of Congo red and isoflavone S.Yet, still need to identify other prion protein binding materials.And still need to identify protein to conformational change, particularly special high affinity bond material with the protein of disease association.These reagent will be used to develop possible diagnostic kit, separate and the multi-form albumen of purification, be used for removing the infectiousness form of this disease from treatment reagent, biological product, vaccine and food, and be used for the treatment of.

Summary of the invention

The method that the invention provides the material that is incorporated into prion protein and use prion protein binding materials (after this being " bond material ").In some embodiments, bond material is a polymer beads, chromatographic grade resin preferably, and it can optionally and specifically combine with the Protein virus analyte.In other embodiment, bond material be special and optionally with the bonded inorganic material of prion protein sample.Bond material can comprise cell prion protein (PrPc), infectious prion albumen (PrPsc) and reorganization prion protein (PrPr) in conjunction with one or more forms of prion protein.Comprise the combined material combination of Protein virus of people and hamster from different plant species.The present invention also provides the compositions that contains the bond material that exists on the holder as chromatographic column.

Bond material is used at sample such as biofluid or environmental sample or detection from sample, combination, separation, removal, eliminating, extraction or separation prion protein.Bond material is used for removing the prion protein of sample form of ownership, perhaps can optionally choose the prion protein that detects or remove single form.Therefore bond material can be used at sample difference infectiousness and non-infectious prion protein from the patient, and the patient comprises the patient of people TSEs and the animal that itching disease, BSE and CWD are arranged.In one embodiment, use bond material described here from biofluid, to remove one or more prion proteins, subsequently biofluid purification or that depollute is used or turned back in people or the animal.Can use hemodialysis technology in this embodiment.Bond material also can be used for detecting one or more prion proteins at sample.

Another aspect of the present invention provides the method for identifying other bond material, especially proteic conformational change form is had specific bond material, and some of them albumen relates to the advancing of disease process.

By following detailed description and embodiment preferred, further feature of the present invention and advantage will be conspicuous.

Description of drawings

Fig. 1 is the photo of Western trace, has shown the combination of the endogenous PrPc among prion protein binding materials and the human plasma sample and suitable contrast.

Fig. 2 is the photo of Western trace, has shown the combination of the PrPsc in prion protein binding materials and the itching disease brain tissue homogenate and suitable contrast.

Fig. 3 is the photo of Western trace, has illustrated that prion protein binding materials catches PrPc and suitable contrast in containing human serum albumin's sample.

Fig. 4 is the photo of Western trace, has illustrated to adopt the resin that contains amino functional group to being incorporated into the removal effect of the PrPres among the human serum albumin.

The specific embodiment

In this method of having described prion protein binding materials and having used prion protein binding materials.Bond material is a polymeric material, as chromatography with resin or pearl, perhaps inorganic material, as aluminium oxide, its can with prion protein specifically and affinity combine.Polymeric material contains one or more following functional group: electronegative part; The part of positively charged; Uncharged part and hydrophobic part.Preferably, the polymer scale condensation material has the functional group that is incorporated into methacrylic acid or polymethylacrylic acid matrix scaffold (matrix backbone).

Prion protein in bond material and the sample forms complex, can from the method for sample or detection sample, combination, separation, removal, eliminating, extraction or separation prion protein, use, wherein the tissue of this sample such as people or animal origin, organ or biofluid or environmental sample.The present invention also provides in people or animal, perhaps the method for diagnosis or monitoring prion disease in its tissue, organ or the biofluid.For example, bond material described here can be by measuring biological sample such as whole blood, the compositions that derives from blood or component, cell, serum, blood plasma, blood plasma derivant, cerebrospinal fluid, urine, tear, tonsil, brain, vermiform appendix and other, thereby be used to detect or diagnose pathology such as CJD, vCJD, GSS, mortality insomnia, itching disease, BSE and CWD and other TSEs.Before blood, tissue or organ transplantation, it is conspicuous that the prion protein in animal or the individuality is infected the importance of measuring.Bond material can be removed prion protein especially from sample or biofluid such as whole blood, blood constitutent, serum, blood plasma, blood plasma derivant and analog.Under biofluid was propagated animal or people's situation to other, as in blood transfusion or use in the blood products as thrombin, the removal of prion protein was very important.All multi-form prion proteins can be removed or detect to bond material from sample, perhaps the prion protein that removes or detect single form can be optionally chosen, and infectiousness and non-infectious prion protein in the sample can be therefore distinguished.

Definition

Be meant " one or more " in the term " " of this application, the definition of " a kind of " and " being somebody's turn to do ", and comprise plural number, unless in context, think inappropriate.

Term " 3F4 antibody " is meant the native form for PrPc, rather than natural PrPsc or PrPres, has specific monoclonal antibody.This antibody has specificity for the hamster of denatured form and people PrPc, PrPsc and PrPres.

As applied at this, term " derives from the compositions of blood ", " blood constitutent " and " blood compositions " can be used mutually alternately, and be intended to comprise whole blood, the erythrocyte concentrate, blood plasma, serum, enrichment or lack hematoblastic part, platelet concentrate, leukocyte, blood plasma precipitate, blood plasma fractional precipitation thing and supernatant, immunoglobulin preparation comprises IgA, IgE, IgG and IgM, the coagulation factor concentrate of purification, fibrinogen concentrate, blood plasma classification intermedium, albumin preparation perhaps derives from other different material of people or animal blood.These terms also comprise by common methods different in the present technique field such as ion exchange, affine, gel sees through and/or hydrophobic chromatography or by the albumen that derives from blood with ethanol or Polyethylene Glycol differential precipitation purification.

Term " PrPc " is meant natural prion protein molecule, and it is natural and be expressed in widely in the mammiferous body.Its structure height is conservative and uncorrelated with morbid state.

Term " PrPsc " is meant the conformational change form of PrPc molecule, those skilled in the art think that this molecule comprises that with disease such as the TSE/ prion protein vCJD, CJD, Kuru disease, fatal insomnia, GSS, itching disease, BSE, CWD and other TSEs are relevant, comprise catch with laboratory animal in rare TSEs.PrPsc has and the aminoacid sequence normal, that cell PrPc is identical, but wherein a part of α spiral has become βZhe Die, and this transformation is relevant with morbid state.

Term " PrPres " is meant that molecular weight is the proteic protease resistant derivant of PrPsc of 27-30kDa, and it still exists after E.C. 3.4.21.64 (PK) part digests PrPsc.

Term " PrPr " is meant the prion protein of expressing by recombinant technique.

Term " PrP " is meant the general name of prion protein.

Term " pearl " is meant solid phase particles or microgranule, and it can association reaction group or binding constituents.Pearl has irregular shape, include sphere, ellipse, shaft-like or or even the shape at band angle, these all belong to the scope of this term.

Term " resin " is meant polymerisation medium.

Chemical compound or the molecule formed by several less, repeated chemistry or construction unit (monomer) described at the term " polymerization " of this application.

Sample

Term " sample " in this application is meant any solution, suspension, extract, compositions, preparation, product, component, tissue, organ, cell or other material object, and they contact with prion protein binding materials according to the method for some aspect of the present invention and embodiment.According to some aspect of the present invention and embodiment, sample includes but not limited to, biological sample, food, environmental sample or water sample.Biological sample includes but not limited to: the sample that derives from blood; Derive from the sample of brain; Body fluid, such as but not limited to, blood, blood plasma, serum, cerebrospinal fluid, urine, saliva, milk, pipe liquid, tear or seminal fluid; Bio-extract is as collagen extract, body of gland extract or tissue homogenate thing or extract.Biological sample derives from people or animal, includes but not limited to cattle, sheep, pig, horse, Mus or Cervidae (Cervidae) animal.The sample that derives from blood comprises, but be not limited to platelet concentrate, plasma protein preparation, immunoglobulin preparation, fibrinogen preparation, factor XI, plasma thromboplastin antecedent II preparation, thrombin preparation, factor VIII formulations, von Willebrand factor formulations, protein C formulations or activatory protein C formulations.Sample according to some aspect of the present invention and embodiment also includes, but are not limited to pharmaceutical composition, therapeutic agent compositions, cosmetic composition and product, food or food or nutritious tonifying compositions.The example of food samples includes, but are not limited to gelatin, fruit jelly, milk, milk product, collagen or infant formula.

According to some aspect of the present invention and embodiment preferred, sample comprises that also containing difference plants proteic protein solutions, includes but not limited to people or animal serum albumin.For example, sample includes but not limited to, contains human serum albumin's therapeutic agent product; The serum albumin preparations of people or animal; Perhaps contain people or animal serum albumin preparation as stabilizing agent.According to some embodiment preferred of the present invention, sample also can contain concentration up to the people of about 50% (w/v) or the serum albumin of animal, and perhaps concentration is from about 1% to about 50%, and perhaps from about 5% to about 25%.In one aspect, in a preferred embodiment of the invention, the present invention unexpectedly and advantageously can be from having the proteic sample of high concentration or removing therein, separate or in conjunction with prion protein, especially from blood protein, as serum albumin.

Environmental sample includes but not limited to soil, sewage or water, Tathagata from the brook, the water or the recuperation water of river, aquifer, well, water treatment facilities.

Sample includes but not limited to, fluid sample, solid sample or colloid sample.Solid sample can extract with liquid phase solvent, organic solvent or critical fluids and obtain, and the supernatant that obtains can be in contact with one another with bond material.The example of solid sample comprises, but be not limited to derive from the product of animal, particularly those are exposed to the sample of the reagent of propagating prion protein, as derive from bone meal, cerebral tissue, cornea tissue, Excreta, bone meal, beef side-product, sheep, sheep side-product, deer and elk and deer and elk side-product and other animal of cattle and derive from the side-product of other animal.

Material

Peptide or the polypeptide that derives from prion protein is provided at this bond material that provides, perhaps complete prion protein molecule, they can be used for various separable programming, include but not limited to chromatography, such as but not limited to, thin layer chromatography, column chromatography and chromatography in batches; Solid support and membrance separation; Reactor separates; Magnetic Isolation; Immunity separates; And colloid separates.In a preferred embodiment, in pillar, comprise bond material, add sample as chromatographic column, and make its by pillar so that the prion protein in the sample is incorporated on the bond material, and be retained in the pillar.Other composition in the sample passes through pillar, and can collect.The application that will be appreciated that bond material described here is not limited to chromatography or column chromatography in batches.Envisioned the various structure, modification and the variation that are used in conjunction with the application of the bond material of prion protein, and this also belongs to scope of the present invention.Such variation and modification include, but are not limited to: batch process, continuous process; Moving bed chromatography process; Low pressure, medium-pressure or high pressure process; Perhaps little, in or large scale process.In selectable embodiment, bond material can be positioned on the film, on the fiber, on the pearl, inject non-woven screen cloth, and coated fiber is included among the filter housings, or the like.

Inorganic component

In first embodiment, bond material contains inorganic compound or component, such as but not limited to, aluminum or silicon oxide.Preferably, aluminum is aluminium oxide, and silicon oxide is a pyrogenic silica.Most preferably, inorganic compound is Al ₂O ₃Perhaps SiO ₂These bond materials can provide with different forms, include but not limited to pearl or resin.Bond material can be used for a lot of different separation processes, and may be arranged in or join chromatographic column, film or any suitable separate apparatus and instrument, perhaps can be used for batch process, perhaps under the condition that allow to form prion protein binding materials and prion protein, make sample contact and be used for any separation process with material.The bond material that contains inorganic compound can comprise a plurality of functional groups.Functional group is hydrophilic, as positively charged, electronegative, neutral or neutrality, hydrophobic, amphoteric or their combination.Below describe special functional group in detail.Should be understood that functional group can intrinsic existence in inorganic compound, perhaps makes inorganic compound comprise functional group by modification.Functional group comprises organic and inorganic functional group.

Polymers compositions

In second embodiment, bond material comprises polymeric material or component, and preferably includes polymeric matrix, is also referred to as the polymeric matrix skeleton simultaneously.Randomly, one or more functional group is incorporated in the polymeric matrix.In preferred embodiments, polymeric material is a resin, preferably, and chromatographic resin.The polymerizable polymer matrix scaffold is the methacrylic acid skeleton preferably,, but is not limited to commercially available TSK as the following stated ^TM, and TOYOPEARL ^TMPerhaps FRACTOGEL ^TMResin (Tosoh Bioscience, Montgomeryville, PA).These include, but not limited to positively charged, electronegative, uncharged, hydrophobic function group or their combination.Particularly preferred functional group has a more detailed description following.

Bond material can be any form, perhaps can make, shape, make in form, the holder that perhaps is used for any surface of solids, comprise, but be not limited to, pearl, film, tube, filter, dip rod, microtitration plate, test tube, pressed powder, casting or extrusion molding dies, screen cloth, the magnetic bead composite, perhaps any other solid material of the following material bag of usefulness quilt is as polyethylene, polypropylene, poly-(4-methyl butene), polystyrene, polyacrylate, polyethylene terephthalate, artificial silk, nylon, poly-(vinyl butyrate), Polyvinylidene Difluoride (PVDF), silicone, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and analog.Selectively, can use the material that forms gel, as albumen (as gelatin), lipopolysaccharide, silicate, agarose and polyacrylamide.The polymer that forms a plurality of waters also is fit to, as glucosan, poly-alkylene ethylene glycol or surfactant, as the alkylammonium salt and the analog of phospholipid, long-chain (12-24 carbon atom).Bond material randomly is distributed in these components.

Bond material is granule, microgranule or pearl form particularly preferably.The microgranule bond material preferably has the scope of size from about 1 μ m to 500 μ m, more preferably is granule or the pearl from about 20 μ m to 150 μ m.

Functional group

Prion protein binding materials according to some aspect of the present invention and embodiment comprises functional group.Term " functional group " in this application is meant chemical group, subunit or substructure, and they give chemistry, physics or physicochemical characteristic to molecule or material.Functional group in this explanation includes, but not limited to hydrophilic functional group, as positively charged, electronegative or uncharged or neutral or hydrophobic functional group.Also envisioned amphiphatic molecule or polyfunctional functional group, and they also belong to scope of the present invention.Functional group comprises organic and inorganic functional group.Preferred functional group contains amine, phenyl or sulphite group.Preferred amido is primary amine, secondary amine, tertiary amine or quaternary ammonium ion, as dimethyl aminoethyl (DMAE) or trimethyl amino-ethyl (TMAE).Other illustrative functional group includes, but are not limited to :-CH ₂-CHOH-CH ₂NH ₂-C ₆H ₅-(CH ₂) ₃-CH ₃-CH ₂-CH ₂-N ⁺H (C ₂H ₅) ₂-SO ₂-CH ₂-CF ₃-CH ₂-CH ₂-N ⁺H (CH ₃) ₂-CH ₂-CH ₂-N ⁺(CH ₃) ₃-SO ₃ ^2-Other available functional groups comprise sulfonyl and 2,2,2-HFC-143a sulfonyl (tresyl).

Although do not want to be subject to this narration, should expect that prion protein has three kinds of different calmodulin binding domain CaMs, they combine with the functional group of positively charged, electronegative functional group and hydrophobic functional group respectively.Therefore, use one or several bond materials, the functional group that each comprises one or more type is used for increasing and/or identifies or remove more specifically the prion protein of sample.When two of application or more a plurality of bond material, sample contacts simultaneously with two kinds or more of bond materials, perhaps one by one, perhaps contacts with any order.Therefore, bond material preferably is made up of two kinds or more kinds of bond material, each or contain the functional group of positively charged, electronegative functional group, uncharged functional group, perhaps contain hydrophobic functional group.When bond material was with particulate form and adopts column chromatography, each dissimilar bond material can be in same pillar or respectively in different pillars.In embodiment preferred more, use three kinds of bond materials, a kind of is the functional group with positively charged, and a kind of is to have electronegative functional group, and a kind of is to have hydrophobic functional group.

As applied at this, term " functional group of positively charged " is meant the chemical constituent that has clean positive charge.The indefiniteness example of positive charge functional group comprises, contains amino group, as primary amine, diethylamino ethyl, dimethyl aminoethyl, trimethyl amino-ethyl and level Four amino group.Term " electronegative functional group " is meant the chemical constituent that has net negative charge at this.Term " uncharged functional group " is meant neutral or uncharged any chemical constituent at this.The non-limiting example of electronegative functional group comprises the group that contains sulphite.In addition, term " hydrophobic function group " is meant for any group that resistance is arranged with water-soaked, comprises alkyl, aromatic group, siloxanes and fluorizated group.The non-limiting example of hydrophobic function group is the group that contains phenyl and butyl.Term " amphoteric functional group " is meant hydrophobic and hydrophilic group simultaneously.

In some aspect of the present invention and the embodiment, the prion combination material contains positively charged functional group, electronegative functional group and does not have electric charge or neutral functional group, hydrophobic functional group or both have.An example of electronegative functional group is the group that contains sulfurous acid.An example of positively charged functional group is an amino group.An example of uncharged functional group is phenyl or butyl group.The example of hydrophobic function group is phenyl group or butyl group.According to some aspect of the present invention and embodiment, use amino group, comprise that primary amine, secondary amine, tertiary amine or the quaternary ammonium group in the bond material all has unique advantage to prion combination.Yet, using different groups, this depends on specific prion protein, sample, and the condition that sample contacts with bond material is all within the scope of the invention.

Various material can randomly be used for bond material, as thin layer, for example, in order to give bond material different desired characters.For example, albumen bag quilt, for example, gelatin can be used to avoid detection or other similar characteristic of non-specific bond and enhancing signal.

Surface-functionalized and basic at interval

In preferred embodiments, bond material has different functional groups on their surface.Be to be understood that these functional groups are present in the surface of bond material inherently, perhaps can join the surface of bond material by the present technique field known program of those of skill in the art.It is known connecting various group or the chemical compound technical method in different surfaces, and these methods are described in detail in the list of references.

Functional group is used in conjunction with prion protein, according to method described here, is used to connect other functional group, perhaps be used for any modification of any physics, chemistry or physicochemical characteristics, the characteristic of material, such as but not limited to, its ion characteristic or hydrophobicity.The functional group that is present in preferred bond material surface includes, but not limited to carboxylic acid, aldehyde, amino, cyano group, thiazolinyl, hydroxyl, sulfydryl, epoxy radicals and similar group thereof.

In preferred embodiments, functional group can comprise base at interval.Base is the group that space and distance are provided between material that also is known as substrate or holder and functional group at interval.Base preferably is made up of carbon, nitrogen or oxygen atom at interval.On the one hand, base is used to change better the prion protein of prion protein binding materials in conjunction with attribute at interval.According to some embodiment, the length of base can be 20 atoms of as many as at interval, perhaps 15 atoms of as many as, perhaps 5 to 10 atoms.At interval base preferably but is not limited to be made up of peptide or its mixture of alkyl, Polyethylene Glycol (PEG), carbohydrate group, aminoacid, the peptide that grows to 20 amino acid lengths or from 1 to 10 amino acid length.Most preferably, the basic at interval combination that contains alkyl and PEG.

Commercially available chromatographic resin

Preferably, bond material is one or more following commercially available chromatographic resin: FRACTOGEL ^TMEMD; TOYOPEARL ^TMAmino, Butyl, Phenyl or AF-Tresyl; Perhaps TSK-GEL ^TMAmino, Phenyl or DEAE resin.More preferably, bond material includes, but are not limited to: FRACTOGEL ^TMEMD TMAE, FRACTOGEL ^TMEMD SO ₃ ^2-, FRACTOGEL ^TMEMD DMAE, Toyopearl ^TMAmino, TSK-GEL ^TMAmino, TSK-GEL ^TMPhenyl, TSK-GEL ^TMDEAE, TOYOPEARL ^TMButyl, TOYOPEARL ^TMPhenyl, aluminium oxide, TOYOPEARL ^TMAF-Tresyl, and tripoli resin.In the most preferred embodiment, bond material is TOYOPEARL ^TMAmino, TSK-GEL ^TMAmino, TSK-GEL ^TMPhenyl or FRACTOGEL ^TMEMD SO ₃ ^2-Envisioned and used other commercially available chromatographic resin and holder, comprised inorganic support, it also belongs to scope of the present invention.

In the preferred embodiment of the invention, bond material comprises polymethacrylates, the hydroxyl polymethacrylates, and perhaps AMINO 650 ^TMResin (Tosoh Biosciences), and amino, as primary amine, secondary amine or tertiary amine.Bond material according to embodiment preferred may further include as structural formula O-R-O-CH ₂-CHOH-CH ₂The interval base, wherein the length of R is 1-10 carbon.Bond material can randomly be coated to or patterning is applied on the solid support, as pearl, film or chromatographic resin.

The evaluation of bond material

Except bond material listed above, can identify other bond material according to as described below.The screening bond material is in conjunction with the ability of Protein virus analyte.Term " analyte " or " a plurality of analyte " are meant multiple molecule as used herein, include, but are not limited to protein, polysaccharide and their any aggregation or mixture.Bond material is hatched with the known sample that contains prion protein, removes unconjugated protein, uses conventional method for example the special traget antibody of prion protein to be detected bonded protein.It is suitable bond material that bonded with it bond material of analyte is identified.There are not anti-or two anti-matched groups to be used to remove the material of non-specific bond yet.

In preferred embodiments, certified bond material Protein virus analyte bonded with it is the prion protein of finding in blood that derives from the human or animal or brain sample.More preferably, analyte is at blood or derives from the product of blood and find.Further preferably, the incitant of analyte and human or animal's TSE or TSE is relevant.

Use bond material to remove prion protein

Useful in multiple analysis, preparation and diagnostic application in conjunction with prion protein or the pulsating bond material of prion protein.In some embodiments, bond material contains solid phase, and the perhaps surface of solids exists with the form of pearl or film, can be used for from the sample combination and removes prion protein or peptide.Bond material can contact sample, and biological example liquid causes forming under the condition of prion protein binding materials complex being enough to, and the prion protein in the sample is attached on the bond material.Bond material separates from sample then, thereby removes and the bonded prion protein of the part of sample.The method that use is used for protein-bonded pearl and film is known in technical field, for example at people's such as Baumbach United States Patent (USP) U.S.5,834,318 and PCT/US01/11150 in describe.

In some embodiments of the present invention, all prion proteins can be removed from sample basically.The meaning that " owns basically " is that the concentration of prion protein significantly reduces.In other words, all samples or sample segment are transferred to healthy patients and can only bring the infectious risk of Protein virus that acceptable is low under the public health guilding principle on one's body.Use single bond material or multiple bond material, all prion proteins can simultaneously or be removed from sample continuously basically.When using multiple bond material, preferably, as described above, use two kinds or more kinds of bond material, every kind contains positive charge functional group, negative charge functional group or hydrophobic function group.In a more preferred embodiment, use two or more bond materials, every kind contains negative charge functional group or hydrophobic function group.The sample adjoining land contacts with two or more bond materials with any order.In preferred embodiments, use three kinds of bond materials, each contains a kind of positive charge functional group, negative charge functional group and hydrophobic function group.

In other embodiment, have only specific Protein virus material from sample, to remove.For example, have only infectious prion (PrPsc) from sample, to remove or have only non-infectious Protein virus (PrPc) from sample, to remove.An important discovery described here is multiple evaluation with bond material of different prion-specifics.Table 4 is depicted as several bond materials and they to hamster and people, the specificity of non-infectious and infectious prion.The preferred combination material that selectivity is removed people PrPsc contains amino group, for example is included in Toyopearl ^TMAmino-650M or TSK-GEL ^TMAmino group in-Amino 750C chromatographic column resin or the suitable thing of its function perhaps contains phenyl group, for example at TSK-GEL ^TMThe phenyl group that contains in the suitable thing of Phenyl-5PW or function.

Preferably, bond material is to be filled in for example pearl in the chromatographic column of pillar.Then sample solution, homogenate or suspension or because gravity or since pressure by pillar, for example high pressure liquid chromatography pillar.Prion protein in the sample will be as on the bond material that is attached in the pillar described here.Collection is removed the level that Protein virus pollutes or reduce at least the Protein virus material by the sample of pillar.

In case sample is by pillar, the prion protein of elution of bound, and its collection is used for analyzing, perhaps if necessary, be used for diagnosis or indication purpose.If prion protein is removed in expectation from bond material, the mobile phase of pillar can at first become removes weak buffer in conjunction with impurity, thus the washing pillar.Then by boiling or containing for example sarcosyl detergent (sarcosyl of strong detergent by adding; Shelton Scientific-IBI; Shelton; CT) or sodium lauryl sulphate (SDS); chaotropic agent is guanidine hydrochloride for example; or the reagent of low pH acetic acid for example, perhaps the chemical modification by binding partner for example the acetylation of amino group remove prion protein from pillar.

Include, but are not limited to whole blood, the compositions that derives from blood or component, serum, cerebrospinal fluid, urine, saliva, milk, intestinal juice, tear, seminal fluid or human or animal's brain source component from the example of the biological sample of wherein removing Protein virus.Other biological sample comprises that those contain the biological sample of collagen or body of gland extract.In one embodiment, Protein virus is removed from human or animal's blood by the hemodialysis ring that use contains one or more bond materials described here.In this embodiment, blood is removed from the human or animal, directly inject the equipment that contains one or more bond materials described here, wherein to remove from blood be because they are attached on the bond material to prion protein, and the blood that reduces of the blood of prion-free or Protein virus directly imports and is back in the human or animal body then.

Prion protein also can use bond material described here from (perhaps be used for animal or be used for people's consumption) biological sample for example food remove.For example, sample contain derive from animal or from the animal material that animal obtains, include, but are not limited to cattle, sheep, pig, horse, Mus, hamster or animal in deer family.Alternately, specimen material can be meant the people; Niu Ke; Sheep section; Suidae; Equine; Muridae for example derives from Mus and hamster; With the material that derives from Cervidae, for example deer and elk.Prion protein can include, but are not limited to gelatin, fruit jelly, milk, collagen and infant formula from the material of wherein removing that derives from animal according to some aspect of the present invention and the related method of embodiment.From wherein also including, but are not limited to according to the sample of removing, component, food or components of cosmetics are added in medicine component, treatment component, nutrition to prion protein according to the related method of some aspect of the present invention and embodiment.

According to embodiment preferred, sample is a protein solution, contains multiple proteins, includes, but are not limited to human or animal's serum albumin.For example, sample can be, but be not limited to, contain plasma protein preparation, immunoglobulin preparation, fibrinogen preparation, Hageman factor I preparation, thrombin preparation, blood coagulation factor VIII preparation, Feng von Willebrand NiaState (von Willebrand factor preparation), protein C preparation, activated protein c preparation or aforesaid any combination preparation or the change of human serum albumin as stabilizing agent; The treatment product that contains the human serum albumin; Human or animal's serum albumin preparations; And contain the diluted protein preparation of human or animal's serum albumin as stabilizing agent.According to preferred embodiment, sample contains the sero-abluminous concentration of human or animal up to about 50% (w/v), perhaps from about 1% to about 50%, and perhaps from about 5% to about 25%.In one aspect, in preferred embodiments, the present invention is unexpectedly and advantageously from having the protein of high concentration, particularly haemproteins, in for example sero-abluminous sample or remove therein, separate or in conjunction with prion protein.

Bond material described here also is used for removing prion protein from environmental sample as deriving from such as the water of streams, river, cistern, well, water treatment facilities or pastime water.

Use bond material to detect Protein virus

Bond material described here also in detection of biological or environmental sample, have prion protein or peptide or quantize prion protein or the method for peptide in use.The biological sample that detects prion protein therein comprises, but be not limited to the extract of whole blood, the compositions that derives from blood or component, serum, cerebrospinal fluid, urine, saliva, milk, intestinal juice, tear, seminal fluid, the compositions that derives from brain, feces or collagen or homogenate, body of gland, tissue (for example tonsil or caecum) or organ.Qualitative and quantitative detection method all is contemplated to, and all of the present invention aspect some and in the scope of embodiment.

About the removal of prion protein, bond material also is used for detecting the existence as the material prion protein that derives from animal of food as described above.For testing goal, term " derives from the material of animal " and is meant material described above and the material that contains the animal parts, for example muscle, connective tissue or organ-tissue.The material that derives from animal further includes, but are not limited to, bone meal, beef, beef side-product, sheep, sheep side-product, deer, deer side-product, Carnis Sus domestica, Carnis Sus domestica side-product, sausage, Petaso and infant food.

Bond material described here also is used for the testing environment sample, for example the prion protein in those environmental samples described above and the soil extract thing.

Owing to find multiple bond material, so that bond material is distinguished in simple sample or between the sample in the method for infectiousness and non-infectious Protein virus is useful with different prion combination characteristics.Therefore, provide the method that is used to diagnose and predict human or animal's prion disease.Prion disease includes, but are not limited to, propagable spongiform encephalopathy (TSEs), and itching disease for example, this disease can influence sheep and goat; Mad cow disease (BSE) can influence cattle; Propagable mink encephalopathy, the chronic Slim (CWD) of spongiform encephalopathy of cat and mule deer, white-tailed deer, mule deer and elk; Kuru, cortex-Basal ganglia-spinal cord degeneration syndrome (CJD), Ge-Shi-Sha syndrome (GSS), the cortex-Basal ganglia of mortality hyposomnia and variation-spinal cord degeneration syndrome (vCJD), these diseases all can influence the mankind.

In one embodiment, make sample, use method described below to detect bonded PrPsc Protein virus by PrPsc comparison PrPc is had the more bond material of high specific.Make identical sample by PrPc comparison PrPsc is had the more bond material of high specific then, use method described below to detect bonded PrPc.Table 4 provides the specificity of several bond materials in conjunction with PrPc and PrPsc.Preferably, the selectivity bond material that detects people PrPsc contains and is similar to TOYOPEARL ^TMTSK-GEL ^TM-Amino 750C Amino-650M or TSK-GEL ^TMThe amino group that contains in-Amino 750C the chemical compound perhaps contains and is similar to TSK-GEL ^TM(all resins are from Tosoh Biosciences, Montgomeryville, the phenyl that contains in PA) for Phenyl-5PW.

When the Protein virus that uses in the method test sample that this provides, sample contacts under the condition that is enough to cause formation complex between prion protein and the bond material with bond material.Detect complex with conventional method then, thus the existence of Protein virus in the test sample.For example, bond material (first part) can carry out labelling with detectable label.As alternative example, complex can pass through labelling second part, for example antibody or other protein combine second part of labelling and sample existing under the condition of bond material, certification mark second part-Protein virus-bond material complex detect.Second part can be covalently or non-covalently is incorporated into Protein virus.Known have multiple label and combination technology, and reported in science and patent documentation widely.In one embodiment, second part is labeled in the process of producing.Suitable label comprises radioactive nucleus thuja acid, enzyme, substrate, cofactor, inhibitor, fluorescent component, chemiluminescent component, magnetic-particle and analog.

Be incorporated into the prion protein of prion protein binding materials, perhaps qualitative the and quantitative detection method of prion protein-prion combination material composite is included in the scope of some aspect of the present invention and embodiment.The prion combination material of formation complex can be filled into or make pillar, film or filter, perhaps is attached to or makes or be fixed on the solid support.Qualitative and detect bonded prion protein quantitatively, and the method that discharges from the prion combination material subsequently is also included within the scope of some aspect of the present invention and embodiment.

Detection can be carried out with any method, comprise immunoblotting, Western analysis, gel mobility shift assay, radioactivity or bioluminescence marker thing tracer method, nuclear magnetic resonance, NMR, electron paramagnetic resonance, arrhea spectrographic method, column chromatography, capillary electrophoresis or other based on size or electric charge, or the molecule tracing method of both variations.Before the detection, second part-Protein virus complex can or can not separate with bond material.Other determination and analysis method includes, but are not limited to, liposome immunoassay (LIAs), and this method is used the liposome that is designed in conjunction with special molecular (as second part), and discharges coated reagent or label.The chemicals of Shi Fanging detects according to standard technique then.

The non-radioactive marker adds with round-about way usually.Usually, second ligand molecular (as, biotin) be covalently bonded on the bond material (first part).Second part is incorporated into the 3rd part (as, chain bacterium antibiotin) molecule then, and the 3rd ligand molecular or inherent the detection perhaps are covalently bonded in signaling system, as detectable enzyme, fluorescent chemicals or chemiluminescence compound.Can use the multiple second and the 3rd part.When second part has the 3rd natural part, for example, biotin, thyroxine and hydrocortisone, second part can be used for combining with the 3rd part labelling, natural generation.Alternately, any hapten or antigen compound can with antibodies.

But the particular marker or the detection moiety that are used to detect bond material-Protein virus complex are not crucial.But detection moiety can be any material with detectable physics or chemical characteristic.Such detectable has obtained good development, and usually, any label that uses in these methods can apply in this method.So label can be any component that can be detected by spectroscope, photochemical, biochemical, immunochemical, electronics, optical or chemical method.Useful label comprise fluorescent dye (as, Fluorescein isothiocyanate, texas Red, rhodamine or the like), radioactive marker (as, ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzyme (as, LacZ (beta galactosidase), CAT (chloramphenicol acetyltransferase), horseradish peroxidase, alkali phosphatase, and generally in EIA (enzyme immunoassay (EIA)) or ELISA (Enzyme Linked Immunoadsorbent Assay), be used as other enzyme that can detect enzyme and colorimetric marker such as gold colloidal or coloured glass or plastics (as, polystyrene, polypropylene, latex, or the like) pearl.Label can directly or indirectly be connected on the component of wanting to analyze according to method well-known in the art.As shown above, many labels can use, and needed sensitivity is depended in the selection of label, the link coupled easiness of chemical compound, stability requirement, the equipment that can obtain and garbage disposal regulation.

Second part also can be directly connected to signal and produce on the chemical compound, as, by linking to each other with enzyme or fluorophor.The enzyme first-selection of the interested thing that serves as a mark is a hydrolytic enzyme, particularly phosphate, esterase and glycosidase or oxidoreductase, particularly peroxidase.Fluorescent chemicals includes, but are not limited to, luciferin and derivant thereof, and rhodamine and derivant thereof, dansyl, umbelliferone, or the like.Chemiluminescence compound comprises luciferin and 2, and 3-dihydro-2 diketone is as luminol.

The method of the known certification mark thing of those skilled in the art.So, for example, be that detection method includes, but are not limited under the situation of radioactive marker at label, do autoradiography with scintillation counter or photographic film.When label is fluorescent marker, can passes through optical excitation fluorescence, and detect resulting fluorescence and detect with suitable wavelength, as passing through microscope, range estimation, by photographic film, by use electron detector such as charge-coupled image sensor (CCDs) or photomultiplier tube, and similar approach.Similarly, the enzyme labelling thing can be by providing the substrate of suitable enzyme, and detect the product that obtains and detect.At last, single colorimetric marker can detect by observing the color relevant with label simply.So in multiple dipping was analyzed, the conjugation gold was purple usually, multiple conjugation pearl is the color of pearl simultaneously.

Bond material of the present invention also can be used for removing or detect prion protein or the peptide from the solid sample material extraction to solution.For example, solid sample can use aqueous solution, organic solvent or intermediary liquid to extract, and the supernatant that obtains can contact with bond material.The example of solid sample includes, but are not limited to, the product of animal origin, and particularly those are exposed to the product that infects Protein virus, as, derive from the bone meal of cattle.In some embodiments, bond material can be used for detecting the prion protein that soil exists.Other solid sample includes, but are not limited to, the side-product of cerebral tissue, cornea tissue, fecal matter, bone meal, cattle side-product, sheep, sheep side-product, deer and elk, deer and elk and the product of other animal or animal origin.

Alternately, Protein virus or prion combination material composite can use E.C. 3.4.21.64 (PK) to handle.PrPc is extremely sensitive to PK, and PrPsc is formed PrPres by part digestion simultaneously.PrPres molecule itself has the height resistance to proteolysis, so PK handles will digest PrPc, and will be converted into PrPres to the PrPsc of PK sensitivity.After removing PK, PrPres can degeneration, is detected by antibody such as 3F4.

In another embodiment, according to the present invention, bond material can be used for the PrPsc of PrPc is selected to concentrate.

It is quantitative that bond material is used for Protein virus

Bond material-Protein virus complex or alternately, the antibody of Protein virus or bond material-Protein virus complex can detect by any method in the known several different methods of those skilled in the art and quantitatively.These methods including, but not limited to, analytical biochemistry method such as spectrophotography, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), high diffusion chromatography (hyperdiffusionchromatography) and similar approach, and panimmunity method, as but be not limited to liquid or gel precipitation reaction, immunodiffusion (single or two), immunoelectrophoresis, radioimmunoassay, RIA (RIAs), Enzyme Linked Immunoadsorbent Assay (ELISAs), immunofluorescence analysis and similar approach.

Reduce non-specific binding

When using solid support to assign to prion protein in the test sample as analysis bank, those skilled in the art should be appreciated that common expectation reduces the non-specific bond to solid support.Those skilled in the art are familiar with reducing the method for so non-specific bond.Usually, this relates to protein component solid support is wrapped quilt.Especially, protein component can be widely used as cattle and people's serum albumin (BSA) and gelatin.

The present invention will be described in more detail by specific embodiment.Following examples are used to illustrate, and are not by any way invention to be limited or limits.

Embodiment 1

The affirmation of prion combination material

Adopt normal hamster brain homogenate liquid, as described below, use NBT/BCIP chromophore (nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate-p-toluene amine salt), in conjunction with experiment, check 80 kinds of polymer or inorganic particulate on the utilization Protein virus pearl.In conjunction with the results are shown in Table 1, wherein "-" expression does not have combination, "+" to represent positive combination."+" in the particle grade is many more, and observed combination is just strong more.Having at least, 12 kinds of particles of " ++ " need further assessment.Table 2 has been summed up the ability of 12 kinds of normal hamster prion proteins of particles combination.The amount of high more branch value representation prion combination increases to some extent.

Table 1

Screening is used for bonded polymeric material of prion protein or inorganic particulate

Reference number	Title	Manufacturer	In conjunction with the result
				1	Toyopearl TM Amino-650M	Tosoh Bioscience (Montgomeryville，PA)	+++
1’	Acetylated Amino-650M	Carry out acetylation by North Carolina State University (NCSU), use Toyopearl TM Amino 650M	-
				2	TSK-GEL TM Amino 750C	Tosoh Bioscience	+++
3	Toyopearl TM Epoxy 700EC	Tosoh Bioscience	+
				4	Toyopearl TM AF-Carboxy-650M	Tosoh Bioscience	-
5	Toyopearl TM AF-Heparin-650M	Tosoh Bioscience	+
				6	Amberchrom TM CG-71m	Tosoh Bioscience	-
7	Amberchrom TM CG-300m	Tosoh Bioscience	-
				8	Toyopearl TM HW-40C	Tosoh Bioscience	-
9	Toyopearl TM HW-50F	Tosoh Bioscience	-
				10	Toyopearl TM AF-Chelate-650M	Tosoh Bioscience	-
11	Toyopearl TM DEAE-650M	Tosoh Bioscience	+

12	Toyopearl TM DEAE-650C	Tosoh Bioscience	-
				13	Toyopearl TM Super Q-650M	Tosoh Bioscience	+
14	Toyopearl TM Super Q-650C	Tosoh Bioscience	+
				15	Toyopearl TM QAE-550C	Tosoh Bioscience	-
16	Toyopearl TM CM-650M	Tosoh Bioscience	-
				16’	Toyopearl TM CM-650C	Tosoh Bioscience	-
17	Toyopearl TM SP-650M	Tosoh Bioscience	-
				18	Toyopearl TM SP-550C	Tosoh Bioscience	-
19	TSK-GEL TM Ether-5PW	Tosoh Bioscience	-
				20	TSK-GEL TM Phenyl-5PW	Tosoh Bioscience	+++
21	Toyopearl TM Butyl-650C	Tosoh Bioscience	++
				22	Toyopearl TM Phenyl-650C	Tosoh Bioscience	++
23	Toyopearl TM Hexyl-650C	Tosoh Bioscience	-
				24	TSK-GEL TM DEAE-5PW	Tosoh Bioscience	+++
25	TSK-GEL TM Q-5PW	Tosoh Bioscience	+
				26	Toyopearl TM AF-Tresyl 650M	Tosoh Bioscience	++
27	Amberlite TM XAD-7HP	Supelco (Bellefonte，PA)	-
				28	Amberlite TM XAD-1180	Supelco	-
29	Diaion/sepabeads TM HP 20SS	Supelco	-
				30	Diaion/sepabeads TM SP 207	Supelco	+
31	MCI Gel CHP 20P	Supelco	-
				32	Silica gel grade 7754	Supelco	-
33	Davisil TM Silica gel grade 634	Supelco	-
				34	Davisil TM Silica gel grade 643	Supelco	-

35	Blue rayon trisulfonated (syn:Copper Phthalocyamine)	Supelco	Not test.It is blue rayon fiber.
				36	Amberlite TM IRC-718	Supelco	-
37	Diaion TM CR 20	Supelco	-
				38	Amberlite TM IRA-958	Supelco	-
39	Dowex TM MSA-1	Supelco	-
				40	Amberlite TM IRA-910	Supelco	+
41	Diaion TM PA418	Supelco	-
				42	Fractogel TM EMD DMAE 650(S)	E.Merck (Gibbstown，NJ)	++++
43	Fractogel TM EMD Phenyl 1 650(S)	E.Merck	-
				44	Fractogel TM EMD TMAE 650(S)	E.Merck	+++++
45	Fractogel TM EMD Propyl 650(S)	E.Merck	-
				46	Fractogel TM EMD Amino(M)	E.Merck	-
47	Fractogel TM EMD SO 3 2-650(S)	E.Merck	++
				48	Fractogel TM EMD COO -650(S)	E.Merck	+
49	PMMA Poly(methylmethacrylate)	Bangs Laboratories (Fishers，IN)	-
				50	Aluminum oxide-100+325	Aldrich (Milwaukee，WI)	++
51	Polyethylene	Aldrich	-
				52	Aluminum oxide-100+400	Aldrich	+

53	Poly(styrene/maleic anhydride)	Sigma Chemical Co. (St.Louis，MO)	-
				54	Aminomethylpolystyrene resin	Aldrich	-
55	Aluminum oxide，activated	Aldrich	-
				56	Silica，fumed	Aldrich	++
57	PVDF SOLVAY 1008/1001	Solvay (Aubum Hills，MI)	++ ++ (non-specific) *
				58	PVDF SOLVAY 1015/1001	Solvay	+++(is non-specific) *
59	Silica gel for column，35-70um	Acros (Pittsburgh，PA)	-
				60	Silica gel 60-200mesh	Acros	-
61	PolyStyrene，0.93um	Bangs	Not experiment, emulsion
				62	Bangs Lab Silica，0.20um	Bangs	Not experiment, emulsion
63	Bangs Lab Silica，0.97um	Bangs	Not experiment, emulsion
				64	1，2DAP 1-Epoxy 700EC	Aldrich	-
65	1，3DAP-Epoxy 700EC	Aldrich	-
				66	1，4DAB 2-Epoxy 700EC	Aldrich	-

67	L-Lysine-Epoxy 700EC	Aldrich	+
				68	TETA 3-Epoxy 700EC	Aldrich	+
69	Prometic CG-1083	Prometic BioSciences (Cambridge，UK)	++ ++ (non-specific) *
				70	Prometic CG-1085	Prometic BioSciences	(non-specific) *
71	Prometic CG-1086	Prometic BioSciences	+ (non-specific) *
				72	Prometic CG-1082 (purple)	Prometic BioSciences	+ (non-specific) *
73	Prometic CG-1084 (purple)	Prometic BioSciences	++ (non-specific) *
				74	Prometic CG-1087 (purple)	Prometic BioSciences	+ (non-specific) *
75	Prometic CG-1014 (purple)	Prometic BioSciences	++ (non-specific) *
				78	Prometic CG-1107 (purple)	Prometic BioSciences	++ ++ (non-specific) *

79	Prometic CG-1108 (purple)	Prometic BioSciences	++ ++ (non-specific) *
				76	Ethylenediamine， polymer-bound	Aldrich	-
77	Pharmacia Source 30Q	Pharmacia (Piscataway，NJ)	+
				80	Clear-Base Resin(HCl)	Peptide International (Louisville，KY)	-

¹DAP: diaminopropanes

²DAB: diaminobutane

³TETA: trien

⁴Amberchom ^TMBe Rohm and Haas Company (Philadelphia, registered trade mark PA)

^*Nonspecific: refer to not have the negative control of antibody 3F4, have and the identical signal of signal that detects with antibody 3F4.

Table 2

The polymerization bond material is in conjunction with the ability of general hamster Protein virus (HaPrPc)

Polymer compound	Basic resin	Manufacturer	HaPrPc
				Fractogel TM EMD TMAE 650(S)	PMMA **	E.Merck	5
Fractogel TM EMD SO 3 2- 650(S)	PMMA	E.Merck						2
				Fractogel TM EMD DMAE 650(S)	PMMA	E.Merck			4

Toyopearl TM Amino-650M	PMMA	Tosoh Bioscience		3
					TSK-GEL TM Amino 750C	PMMA	Tosoh Bioscience		3
TSK-GEL TM Phenyl-5PW	PMMA	Tosoh Bioscience		3
					TSK-GEL TM DEAE-5PW	PMMA	Tosoh Bioscience		3
Toyopearl TM Butyl-650C	PMMA	Tosoh Bioscience		2
					Toyopearl TM Phenyl-650C	PMMA	Tosoh Bioscience		2
Aluminum oxide-100+325	Al 2O 3	Aldrich	2
				Aldrich silica，fumed	SiO 2	Aldrich	2
Toyopearl TM AF-Tresyl 650M	PMMA	Tosoh Bioscience						2

^*PMMA: polymeric methacrylate

On the following Protein virus pearl of using NBT/BCIP in conjunction with experiment (prion bindingon-beads test).Obtain normal PrP from 10% logical sequence murine brain homogenate, sample is dissolved in 0.5% sarcosyl (Sarkosyl) (200 μ L 10% to the 4ml cerebral tissue) 30 minutes in room temperature in agitator.Sample is with 14, centrifugal 5 minutes of 000rpm.Abandon supernatant, and it is diluted with required culture medium.With 96 hole microtitration flat board (Cat.No.3075, Becton Dickinson, Franklin Lanes, NJ) and Millipore MultiScreen-DV plate (Cat.No.MADV N6510, Millipore company, Bedford, MA), at first use available from Pierce (Rockford, 1% (W/V) casein IL) with 200 μ L/ holes in 65 ℃ the sealing 1 hour.10 milligrams of (10mg) dried pearls swelling in the 10mM of 1ml PBS pH 7.4 is washed 2 times.Turned letter microtitration plate, every hole add the suspension of 20-30 μ l swelling pearl.The suspension sedimentation discards redundant moisture.

Normal logical sequence Mus brain homogenate thing is diluted, be with the human serum albumin that is diluted in 5% at 1: 10 (Alpha Therapeutic Corp.Los Angeles, CA) in, wherein 5% human serum albumin under 60 ℃ by heat treatment 10 hours.Every hole added the suspension of 150 μ L volumes, with pearl incubated at room 1.5 hours.Discard unconjugated protein solution, add in every hole 100 μ L, be diluted at 1: 4000 3F4 monoclonal antibody in 1% casein (SignetLaboratories, Inc., Dedham, MA).Control wells contains 1% casein of 100 μ l.Pearl is hatched with 3F4, spends the night in 4 ℃ of slight wobble.

Pearl is with the PBS+10 μ M CuCl of 10mM pH 7.4 then ₂ Wash 2 times.With second antibody, (St.Louis MO), diluted in 1% casein with 1: 1000, and added in the hand-hole with the volume in 100 μ l/ holes for #A3688, Sigma promptly to combine the anti-Mus IgG conjugate of alkali phosphatase.The vibration of sample room temperature was hatched 1 hour.All pearls are transferred to Millipore, and (Bedford, MA) the MultiScreen-DV plate washs.The sample PBS+Cu of pH 7.4 ²⁺PBS+Cu is used in+Tween 20 (0.05%) washings 3 times ²⁺Wash 3 times, with 1M NaCl washing 2 times, with the 50mM Tris-HCl+5mM MgCl of pH 9.5 ₂ Wash 2 times.

With 1 step NBT/BCIP substrate mixing, 100 μ l are directly added in each hole, up to obtaining ideal colour developing (brilliant violet).Generally hatched 5-15 minute.(Hercules CA) is cut into suitable shape to filter paper for #1703932, BioRad, and is moistening with distilled water.Join S ﹠amp under the pearl suspension vacuum; (Schleicher-Schuell Bioscience, Keene is in trace hole NH) in S Minifold I Dot-Blot system.The hole water rinse scans the result in the computer.

Embodiment 2

Determining of prion combination material

Use two kinds of different detection systems, carry out the evaluation of prion combination material to become batch mode with logical sequence Mus brain homogenate thing.At first, hatch back pearl is dyeed with target material and detect the amount of prion combination to material.Second method detects the Protein virus amount that exists in the not binding constituents that contains in the effluent, and with SDS-PAGE and western trace washing sample.Being described in detail as follows of every kind of method.

As shown in the following Table 3, Toyopearl ^TMAmino-650M, TSK-GEL ^TMAmino750C and TSK-GEL ^TMPhenyl-5PW provides the most special combination of logical sequence Mus brain PrPc.

Table 3

The result of programmed screening

Polymer beads	By experimental evaluation combination on the pearl of using NBT/BCIP	Estimate combination by Western trace with ECL-plus
			Toyopearl TM Amino-650M	+++	+++
TSK-GEL TM Amino 750C	+++	+++
			TSK-GEL TM Phenyl-5PW	+++	++++
Toyopearl TM Butyl-650C	++	+
			Toyopearl TM Phenyl-650C	++	+
TSK-GEL TM DEAE-5PW	+++	5+ (non-specific)
			Toyopearl TM AF-Tresyl 650M	++	-
Fractogel TM EMD DMAE 650(S)	++++	5+ (non-specific)
			Fractogel TM EMD TMAE 650(S)	+++++	5+ (non-specific)
Fractogel TM EMD SO 3 2-650(S)	++	+++
			Aluminum oxide-100+325	++	+
Aldrich silica，fumed	++	5+ (non-specific)

For detection method on the pearl, with 96 hole microgranule titer plate (Cat.no.3075, Falcon, Becton Dickinson, Franklin Lanes is NJ) with Millipore Multiscreen-DV plate (Cat.no.MADV N6510, Millipore, Bedford MA) sealed 1 hour in 65 ℃ with 200 μ L/ holes with 1% (w/v) casein.Every kind of polymer of 10 milligrams every part are soaked among the 10mM PBS of pH 7.4 of 1mL, wash 2 times.Emptying 96 orifice plates, every hole adds the resin suspension of 20-30 μ L.Resin settled discards redundant solution.Add the normal hamster brain homogenate solution of 150 μ l in the resin to be dissolved among 5% human serum albumin at 1: 10.Mixture incubated at room 1.5 hours.Drain hole adds the 100 μ l 3F4 antibody (1: 4000) that are in 1% casein in every hole, slight shaken overnight is hatched in the refrigerator.Pearl is with the PBS+10 μ M CuCl of 100mM pH 7.4 then ₂ Wash 2 times, (St.Louis MO), slightly vibrates incubated at room 1 hour for Cat no.A3688, Sigma to add the second antibody that combines alkali phosphatase with 100 μ l/ holes then.

The hole that emptying is all is transferred to pearl in the Multi-Screen plate, uses the 10mM PBS+10 μ M CuCl of pH7.4 on this plate ₂+ 0.05%Tween 20 uses 10mM PBS+10 μ M CuCl then with pearl washing 3 times ₂Washing is with 1M NaCl washing 2 times, with the 50mM Tris-HCl+5mM MgCl of pH 9.5 ₂ Wash 2 times.

Pearl that has washed then and 100 μ L NBT/BCIP substrate reactions 5-15 minute are with colour developing.Use S﹠amp; S Minifold I Dot-Blot system transfers to pearl on the filter paper, carries out the mensuration of the color that forms.

For the mensuration of bound fraction not, every kind of pearl sample of 100 μ L spends the night moistening in advance in 4 ℃ with the 10mM PBS of pH 7.4, place microcentrifugal tube.At least after washing 4 times with PBS, shift pearl to Ultrafree-MC 0.45 μ m filter element (UFC30HVNB, Millipore, Bedford, MA) in, wash once more with PBS.Handle 10% hamster brain homogenate liquid (HBH) with 0.5% sarcosyl, diluted with 1: 10 and 1: 20 with PBS.It is added 200 μ l in every kind of pearl sample, vibration was hatched 8 minutes, and then with 10, centrifugal 2 minutes of 000rpm reclaims not bound fraction.The effluent of 26-μ L is placed the 0.7mL microcentrifugal tube, and the Western engram analysis is carried out in-20 ℃ of storages.

Carry out the sample that thaws before the Western trace, add 10 μ L sample buffer (NuPAGE, SDS sample buffer, NP0007, Invitrogen, Carlsbad is CA) with 4 μ L Reducing agent (NuPAGE sample Reducing agent, NP0004, Invitrogen) (DTT, 1M are dissolved in H ₂Among the O).Hatched 10 minutes for solution 90-100 ℃.With sample be added to 15 holes NuPAGE 4-12%Bis-Tris gel (NP0323, Invitrogen) on.For every hole gel, the western engram analysis uses 17 μ L samples, and the protein staining gel uses 14 μ L samples.(MultiMark Multi-Colored Standard, LC5725 Invitrogen) are 5 μ L to the volume of molecular weight marker thing.The Western trace uses pvdf membrane, and 1% casein is as sealer, and 1: 10,000 3F4 was anti-as one, and 1: 3000 the sheep anti-mouse antibody that is connected with horseradish peroxidase (HRP) is anti-as two, and ECL+ is as substrate.Exposure 6 minutes.

Be attached to sample on the bond material, that have high PrP and in effluent, do not produce signal, be worth and be " 5+ ".There is not the value of bonded sample to be "-".The value of other sample is between these two values.

Embodiment 3

Determine the prion protein binding specificity

In general, will place independent disposable pillar by the moist pearl that different bond materials are formed quantitatively.The pillar that contains glass material is little as can only to keep pearl, but even as big as allowing activated solution (challenge solution) flow through.Activated solution is the brain homogenate liquid that contains prion protein, is dissolved in the sarcosyl (Sigma); Being incorporated into erythrocyte concentrates in the blend.More specifically, activated solution contains TSE infectiousness human brain homogenate, infectiousness hamster brain homogenate liquid, non-infectious human brain homogenate or non-infectious hamster brain homogenate liquid.Allow activated solution in the time durations of determining, to pass through the target bond material, collect effluent simultaneously.Wash pearl then, quantitatively transfer to the receiving flask, in order to determine special prion protein combination and non-specific protein bound processing subsequently, from receiving flask, remove the pearl of known quantity like this from the pillar of bead loading.Store effluent and remaining reaction pearl, so that possible analysis later on.

Use the following more detailed method of describing, determine that the combination of seven kinds of prion protein binding materials is active, as describing in the table 4.Bond material is to come classification (rank that shows in conjunction with the bond material of maximum prion protein is 1) in conjunction with the binding ability of normal or infectiousness people or hamster prion protein.For example, Fractogel ^TMEMD TMAE 650 (S) bond material (has methacrylic acid skeleton and functional group-CH ₂-CH ₂-N ⁺(CH ₃) ₃) in conjunction with hamster and people's infectious prion albumen (PrPsc) of maximum, Fractogel ^TMEMD SO ₃ ^2-650 (S) bond material (has methacrylic acid skeleton and functional group-SO ₃ ^2-) in conjunction with the hamster and the normal prion protein of people of maximum.These quantity are to determine by the following method, bonded albumen is discharged from the prion combination material, the protein that electrophoretic separation discharges uses the Western engram analysis to discharge and next proteinic immunocompetence from bond material, adopts the special monoclonal antibody of Protein virus to carry out.Antibody passes through chemiluminescence detection to the combination of prion protein.Carry out quantitatively (antibody that indication combines with the prion protein that is attached on the bond material) by the darkness that compares the electrophoretic band on the film, the contrast band is 2ng, 10ng and 50ng Mus IgG, thus bond material is determined score value.By comparative sample band directly mutually, determine to have the grade of identical fractional bond material.

Table 4

The classification of 11 kinds of polymerizable compounds

After the postsearch screening, based on them and normal hamster Protein virus (HaPrPc), normal person's Protein virus (HuPrPc), the binding ability of infectiousness hamster Protein virus (HaPrPsc) and infectiousness people Protein virus (HuPrPsc) is carried out classification.

Polymer compound and functional group	Base resin	Manufacturer	HaPr Pc	HuP rPc	HaPr Psc	HuP rPsc
							Fractogel TM EMD TMAE 650(S) -CH 2-CH 2-N +(CH 3) 3	PMMA	E.Merck		2	2	1	1
Fractogel TM EMD	PMMA	E.Merck		1	1	10									9

SO 3 2-650(S)-SO 3 2-
							Fractogel TM EMD DMAE 650(S) -CH 2-CH 2-N +H(CH 3) 2	PMMA	E.Merck		3	3	2	7
Toyopearl TM Amino-650M -CH 2-CHOH-CH 2NH 2	PMMA	Tosoh Bioscience		6	10	4									4
							TSK-GEL TM Amino 750C -CH 2-CHOH-CH 2NH 2	PMMA	Tosoh Bioscience		5	9	3	3
TSK-GEL TM Phenyl-5PW-C 6H 5	PMMA	Tosoh Bioscience		7	5	5									2
							TSK-GEL TM DEAE-5PW -CH 2-CH 2-N +H(C 2H 5) 2	PMMA	Tosoh Bioscience		4	7	6	8
Toyopearl TM Butyl-650C -(CH 2) 3-CH 3	PMMA	Tosoh Bioscience		8	4	8									5
							Toyopearl TM Phenyl-650C C 6H 5	PMMA	Tosoh Bioscience		9	6	9	6
Aluminum oxide-100+325	Al 2O 3	Aldrich	10	8	7	10
							Toyopearl TM AF-Tresyl 650M SO 2-CH 2-CF 3	PMMA	Tosoh Bioscience		11	11	11	11

Prepare the no drop of water

By moistening 20% methanol solution of pearl water preparation, the no drop of water of a large amount of preparations.Before using pearl was left standstill 24 hours at least.When the initial amount of the no drop of water when 0.5g is between 2.5g, the pearl serosity of prewetting is transferred in the 50ml plastics conical pipe.Discard unnecessary liquid, add the methanol of 25ml 20%.Sample slightly vibrates or put upside down 30 seconds then.When initial pearl weight exceeds aforementioned range, regulate the volume of methanol, the resin that is less than 0.5g uses 10ml methanol, 2.5 to 4.0g resin use 40ml methanol.Pearl perhaps was deposited to the pipe end up to the full pearl of major part by the about 12-15 of gravitational settling minute.Extract carefully and supernatant discarded solution (comprising chip).With methanol wash once more than, add 20% methanol then, become 1: the 1 pearl serosity of (v/v).Pearl is in 4 ℃ of storages.

Preparation pearl serosity

Shift 440 microlitres (440 μ l) pearl serosity to 15ml plastics conical pipe (each pillar use 220 μ l wet pearl), add 10ml working buffer liquid (20mM citrate buffer solution/140mM NaCl, pH 7.0).Sample slightly vibrates or put upside down 30 seconds.Pearl perhaps was deposited to the pipe end up to the full pearl of major part by the about 12-15 of gravitational settling minute.The careful absorption and supernatant discarded solution (comprising chip).Working buffer liquid is repeated to wash more than 2 times, add the working buffer liquid of capacity, making volume ratio is 1: 1.Sample slightly vibrates once more or put upside down 30 seconds, sedimentation, room temperature standing over night.To keep the volume of working buffer liquid be 1: 1 by adding necessary working buffer liquid, the hydration pearl remained in the buffer in 4 ℃, up to use.

Prepare pre-hydrated pearl

AminoToyopearl ^TMObtain with the form of other pearl, do not need to carry out extra hydration step with the wet pulp liquid in 20% ethanol.Yet, following balance to working buffer liquid.The manufacturer estimates that it is about 72% resin that the commercialization serosity contains percent by volume, and every pillar uses 300 μ L serosity.Shift in pearl to the 15 milliliter plastic tube (for example Falcon), add 10ml working buffer liquid.Sample slightly vibrates or put upside down 30 seconds.Pearl perhaps was deposited to the pipe end up to the full pearl of major part by the about 12-15 of gravitational settling minute.The careful extraction and supernatant discarded solution (comprising material in small, broken bits).Working buffer liquid is repeated flushing at least 2 times, add the working buffer liquid of capacity, making volume ratio is 1: 1.Sample slightly vibrates once more or put upside down 30 seconds, sedimentation, room temperature standing over night.To keep the volume of working buffer liquid be 1: 1 by adding necessary working buffer liquid, the hydration pearl remained in the buffer in 4 ℃, up to use.

Erythrocytic preparation and processing

Adsol with one bag of 110ml ^TM(Baxter, Bloomington IN) are added in about 250-300ml RBC of containing of one bag of about 250ml and passenger leucocytes and the hematoblastic erythrocyte concentrated solution (RBC).The hematocrit that obtains (erythrocyte (RBC) volume/cumulative volume) is approximately 50-55%.Adsol ^TM: CPD (citric acid phosphoric acid dextrose) and RBCs put upside down mixing.Then will (Pall Corporation, East Hills NY), place new bag by Pall leukoreduction filter from following eight hours of the mixture room temperature of collecting.This process has reduced leukocytic amount in the RBC mixture.Filtering out leukocytic RBC can preserve in new bag in 4 ℃ and reach 42 days.The final component of buffer solution mixture is 30.6%Adsol/8.5%CPDv in this preparation: v.Before the use, detect the percentage of hemolysis of RBC preparation, undertaken by the absorbance of supernatant of measuring after centrifugal at the 415nm place.With respect to the haemolysis value that obtains immediately after the preparation, the increment of haemolysis value is not used greater than 2% RBC preparation.

The preparation of brain homogenate liquid and processing

Normal hamster brain homogenate liquid (10%w/v) is prepared according to the method that they set up by the Dr.Robert Rohwer and the colleague of University of Maryland (the University ofMaryland).Prepare equal portions solution with the 1.8ml volume, preserve in-80 ℃ of freezing preservations or liquid nitrogen, up to use.Selectively, homogenate melts and once carries out packing.The every pillar of each experiment uses the brain homogenate liquid of 60 microlitres.

After brain homogenate liquid sample melted, sample placed and wets on ice.The 5% sarcosyl reagent that adds 6.6 μ l then in the equal portions solution of the brain homogenate liquid that per 60 μ l melt (is dissolved in the 0.5g sarcosyl CPD of 9.5ml: (8.5%CPD, 30.6%Adsol and 60.9%PBS v: v: v)) in the Adsol buffer.Sample the wet vibration of vortex on ice 30 minutes, carries out degeneration slightly.Sample is in micro centrifuge 4 ℃ then, and 14, centrifugal 5 minutes of 000rpm.Shift supernatant to new pipe, discard precipitate.Supernatant places and wets on ice then, and is the longest 1 hour.The supernatant that obtains is the 10% brain homogenate liquid that contains 0.5% sarcosyl reagent.The aliquot that obtains is added on the pillar in preparing in back 1 hour, places in the processing procedure and wet on ice.

The hydration pearl is quantitatively transferred on the pillar

On each void column, add 750 μ L0.1%Tween ^TM20 solution.Ethanol (the v: v), it is flow through under gravity that adds 1 milliliter 20% then on each pillar.Add the deionized water of 2 * 1ml degasification again on every pillar,, remove any air that is captured in the frit to wash alcoholic solution off.Use quantitative pipettor, the hydration pearl suspension of 400 μ l is transferred in the pillar.The wet pearl that unnecessary working buffer liquid relies on the gravity current transmission from one meridian to another to shift before importing sample, washs pillar 3 times with 1ml working buffer liquid then.

Preparation RBC blend (activated solution)

With syringe and No. 18 (perhaps more large size) syringe needle, the RBC/ pillar of 540 μ l is placed the polypropylene conical tube.For Adsol ^TMThe layer be located away from top, with pipe with 3, centrifugal 10 minutes of 000rpm.Then toward top Adsol ^TMAdd the brain homogenate liquid that 10% of 60 μ l handled on the layer, thereby minimizing RBC preparation and high concentration are mixed material, i.e. direct contact between brain homogenate liquid and the sarcosyl detergent.Blend is put upside down mixing, places to wet on ice, uses in 4 hours of preparation.Before the post analysis is used, make mixture return to room temperature, kept 10 minutes.

Activated solution is added in the pillar

After all pillars had all been filled the hydration pearl, admixture activation solution very carefully was added to it on pearl with the volume layering of 0.5ml/ post.Make solution pass through pillar by gravity.Total flow time is between about 5 to 20 minutes.

The outflow activated solution of initial 0.5ml is collected in the 2ml cryovial.Add 0.5ml working buffer liquid again in each post, effluent regathers in the identical cryovial.The pillar that pearl is housed is washed 5 times with 1 milliliter of working buffer liquid, and wherein pearl is resuspended continuously by pipetting, to guarantee thoroughly and uniform the washing.Then, according to as described below pearl is reclaimed from post.

The bonded pearl of quantitative recovery

In every post, add 0.75ml working buffer liquid., with the suspension pearl suspension is transferred in the graded tube fast with pipet flushing pillar.The sedimentation in pipe of pearl suspension is removed supernatant and not disturbance bead layer as far as possible.Supernatant is added back in the identical pillar then, above step is repeated 2 times, so that any pearl remaining in the pillar is transferred in the pipe.Allow pearl to pass through gravitational settling 10 minutes then, the bead layer volume in the graduated pipe of record paper.

The preparation be used to analyze in conjunction with pearl

At first, the working buffer liquid horizontal adjustment in every pipe to 1ml, is made the pearl suspension by slight vortex pipe then.Use pipet to shift out the suspension of 500 μ l, transfer to little Eppendorf ^TM(Brinkmann instruments, Westbury is NY) in the microcentrifugal tube.Allow suspension sedimentation 10 minutes, with the volume-adjustment of settled pearl to 100 μ L.Pearl after the centrifugal then transfer is removed supernatant.Prepare pearl equal portions solution immediately, carry out electrophoresis and western engram analysis.

The Quantitative Comparison of the no drop of water and hydration pearl

Volume with the sedimentation pearl is the basic calculation dry weight, and expansion rate is as follows:

Pearl dry weight=settling volume/expansion rate

Expansion rate=hydration pearl volume (μ L)/dry weight (mg)

For Toyopearl ^TM, the wet pearl of 200 μ L of 42.5mg dry weight=in 20% methanol

Expansion rate (in 20% methanol)=200/42.5=4.71

Embodiment 4

The Western engram analysis

Following Western western blot procedure is designed for assessment from reclaiming in the brain homogenate liquor that mixes erythrocyte concentrated solution (RBC) or removing infectiousness or non-infectious prion protein.These programs are used for the sample of acquisition from the pillar prion combination that above embodiment 3 describes is analyzed, and comprise the pearl sample that is exposed to activated solution and flow through the activated solution sample of pillar, and collect these samples.

Usually, the pearl of targeting bond material and the solution reaction that contains Protein virus, sample source perhaps derives from the effluent of these reactions in the pillar binding analysis of such pearl.Come the existence of prion protein in the sample of analyte preparation then by the Western trace.Use resists the fluoroscopic examination that carries out prion protein to the special mouse monoclonal antibody of prion protein as one.With the two anti-immune complexs that detect these Protein viruss that are combined with alkali phosphatase, chemiluminescence reaction is developed then with the X-ray sheet.

The gel electrophoresis of sample formulation

After the pillar analysis of in embodiment 3, describing, preferably carry out following steps immediately.

To the pillar pearl sample of each preparation, the good pearl of the suspension of 100 μ L is mixed with the Invitrogen 2X sample buffer of 100 μ L by eddy current.Equally by the untapped brain homogenate liquid that obtains in the post analysis (normal human brain, sporadic CJD brain, normal hamster brain, sheep itching disease hamster brain, or the like) be mixed with matched group with Invitrogen 2X sample buffer.More specifically, in the 2X sample buffer of 40 μ L, add 20 μ L brain homogenate liquid.

Be prepared as follows Mus IgG contrast equally.By the Mus IgG of the 2.5mg/ml of 20 μ l is mixed with the PBS of 480 μ l, prepare the reference material of every swimming lane 50ng, be equivalent to 100 μ l/ml.This mixture 25 is added in 475 the 2X Invitrogen sample buffer, obtains 5ng/ml solution.For high concentration directly go up sample gel reference material, use this reference material with every swimming lane 10 μ l, provide the 50ng/ swimming lane.The Mus IgG solution of 5 microlitres, 100 μ g/mL is mixed with 495 μ l 2X Invitrogen reduction sample buffer.This has produced the Mus IgG of 1ng/ μ L; Every swimming lane uses this mixture of 10 μ l, obtains 10ng/ swimming lane (high concentration directly goes up sample gel reference material) (10ng/ swimming lane).Also the middle concentration standard thing by the dilution abovementioned steps prepares Mus IgG low concentration and directly goes up sample gel reference material (2ng/ swimming lane), and the middle concentration standard thing of the abovementioned steps i.e. 1ng/ μ L Mus IgG of 50 μ L by will being dissolved in sample-loading buffer mixes with the Invitrogen 2X sample buffer of 200 μ L and obtains (generation 0.2ng/ μ L).Sample 10 μ L are the 2ng/ swimming lane on every hole.Also can be as the preparation Invitrogen SeeBlue Plus2 Pre-Stained molecular weight standard thing of manufacturer's guidance.

All samples in the Invitrogen buffer 90 ℃ the heating 10 minutes.Of short duration then centrifugal sample ,-20 ℃ of overnight storage.The next morning is repeating heating schedule with sample application before the SDS-PAGE gel.

The immunoreation program

After 12%Bis Tris NuPAGE SDS-PAGE gel was with sample on the sample as described above, gel carried out the electroblotting branching program with constant voltage 200V electrophoresis 45 minutes.The film that protein transduction is moved on on it places the square ware of Fisher, uses 25ml Western Breeze sealer (12.5ml water, 5ml diluent A and 7.5ml diluent B) incubated at room 1 hour on shaking table.Discard lock solution.

Film is hatched in the 000 Signet 3F4 one anti-solution that is diluted in the fresh Western Breeze of the 20ml one anti-diluent (14ml water, 4ml diluent A, 2ml diluent B) with 1: 5.One anti-was diluted in the glycerol with 1: 1 in advance, so working dilution is 1: 10, and 000.Film is under freezing conditions hatched on shaking table.

Discard an anti-solution,, use 20ml Western Breeze at every turn film washing 3 times ^TMAntibody wash solution (1.25ml antibody wash solution (16 *) is dissolved in the 18.75ml water) washed on shaking table 10 minutes in room temperature.Then film with AP3 (KPL, Gaithersburg, MD) two anti-hatching, two anti-be with 1: 10,000 is diluted in the solution that obtains in the 20ml Western Breeze one anti-diluent, room temperature was hatched on shaking table 60 minutes.Discard two anti-solution, use WesternBreeze Antibody Wash washing film as described above.Use the 20mM Tris-HCl of 20ml pH 9.8 then, 1mM MgCl ₂Room temperature was with film washing 10 minutes.

The chemiluminescence generating routine

Transfer membrane is soaked in chemical luminous substrate (the CDP Star of 5ml Western Breeze premix to exsiccant pallet ^TMSubstrate, Applied Biosystems, Foster City stirred 5 minutes in CA) gently.Blot film gently with napkin, place protection sheet then.Then film being transferred to from protection sheet in the magazine (does not have intensifying screen), and room temperature kept 30 minutes, and room temperature was exposed to radiography 5 minutes.

Embodiment 5

From human plasma in conjunction with endogenous PrPc

Remove the ability of PrPc in order to show the prion combination resin from human plasma, carry out following experiment, described human plasma is a human plasma endogenous, that do not penetrate PrPc.

For by the prion combination material in conjunction with the effect of endogenous PrPc, use be human plasma undiluted, fresh, that converge.Freezing blended human plasma thaws for 37 ℃, filters by 0.45 μ m filter, and adding sarcosyl to final concentration is 0.05%.As other local description in this description, carry out combining of blood plasma and pillar, and the detection of Protein virus.

Experimental result in conjunction with endogenous PrPc from human plasma is described in Fig. 1.Group A is described in when not having sarcosyl, and (the 1st road is the molecular weight marker thing to the Western trace testing result of prion protein in the pearl eluent; The 2nd road-low concentration Mus IgG; The 3rd road-middle concentration Mus IgG; The 4th road-high concentration Mus IgG; The 5th road-normal human blood platelets; 6-7 road-resin a; 8-9 road-resin b; 10-11 road-Amino 650-M; 12-13 road-acetylation Amino 650).Western trace testing result (the 1st road-molecular weight marker thing of the not bound fraction of sample among the group B description group A; The 2nd road-low concentration Mus IgG; The 3rd road-middle concentration Mus IgG; The 4th road-high concentration Mus IgG; The 5th road-normal hamster brain (nHB); The 6th road-normal human blood platelets, 7-8 road-resin a; 9-10 road-resin b; 11-12 road-Amino 650-M; 13-14 road-acetylation Amino 650).Group C is illustrated in when having sarcosyl, and (the 1st road is the molecular weight marker thing to the Western trace testing result of prion protein; The 2nd road-low concentration Mus IgG; The 3rd road-middle concentration Mus IgG; The 4th road-high concentration Mus IgG; The 5th road-normal hamster brain; The 6th road-human blood platelets, the 7th road-human normal plasma+sarcosyl, 1: 10; The 8th road-resin a; The 9th road-resin a; The 10th road-resin b; The 11st road-resin b; The 12nd road-Amino 650-1; The 13rd road-Amino 650-2).Group D is illustrated in when having sarcosyl, and by the Western trace of prion protein, (the 1st road is the molecular weight marker thing to the result of the not bound fraction in the test set C sample; The 2nd road-low concentration Mus IgG; The 3rd road-middle concentration Mus IgG; The 4th road-high concentration Mus IgG; The 5th road-normal hamster brain; The 6th road-human blood platelets, the 7th road-human normal plasma+sarcosyl; The 8th road-resin a; The 9th road-resin a; The 10th road-resin b; The 11st road-resin b; The 12nd road-Amino 650-1; The 13rd road-Amino 650-2).

With reference to figure 1, group A, the 10th and 11 roads and group C, the 12nd and 13 roads detect PrPc and are attached to Toyopearl ^TMCombination on the Amino 650-M resin; If except that the electric charge on deaminizing, so just can not carry out in conjunction with (result is as group A, shown in the 12nd and 13 roads) by acetylation.

Experimental result has shown resin ability in conjunction with endogenous PrPc from human plasma, thereby provides the resin can be used for removing from the human or animal evidence of PrP sample.

Embodiment 6

From blood component in conjunction with the sept requirement of PrPsc

As shown in Figure 1, Toyopearl ^TMAmino 650-M resin from human plasma in conjunction with endogenous PrPc.At least the part of this resin contains spacerarm or group, is Tosoh ^TMInstitute is proprietary.The research sept is to the bonded importance of PrPsc.

Following filling four (4) is used for determining the Separation of Proteins test kit (PIKSI of absorbent ^TM) pillar (0.5ml/ each): two pillars of each laboratory sample, one is the Toyopearl that lacks sept ^TMAmino 650M, one is the commercial Toyopearl that sept is arranged ^TMAmino 650M resin.Also detect the Toyopearl that does not have sept simultaneously ^TMAmino 650C resin.

Handle 2 milliliters of (2ml) 10% sheep itching disease brain homogenate liquid (SBH) with 0.5% sarcosyl.By 3ml SBH being added in the 297ml working buffer liquid, the supernatant that sarcosyl was handled dilutes (1: 100) with the work buffer, and the supernatant that pillar was handled with such sarcosyl activates.Pillar, activates 2 times by with sample on the flow velocity of 0.5ml/min with the SBH that 10ml is diluted in buffer.Collect effluent, from each pillar, remove resin equal portions thing, with the washing of 10ml working buffer liquid.

With the activated solution in every kind of resin and the buffer to every kind of sample wherein half uses protease K digesting.Sample as described in other place of this paper, carries out the Western trace and detects.(the 1st road is the molecular weight marker thing to the result who obtains as shown in Figure 2; The 2nd road is the SBH-PK that handles with usefulness 0.1% sarcosyl of buffer preparation; The 3rd road is the SBH+PK that handles with usefulness 0.1% sarcosyl of buffer preparation; The 4th road is MWM; The 5th road is Amino 650M (commercial)-PK (1); The 6th road is Amino 650M (commercial)-PK (2); The 7th road is Amino 650M (commercial)+PK (1); The 8th road is Amino650M (commercial)+PK (2); The 9th road is Amino 650M (test)+PK (1); The 10th road is Amino 650M (experiment)+PK (2); The 11st road is Amino 650M (experiment)+PK (1); The 12nd road is Amino 650M (experiment)+PK (2); The 13rd road is Amino650C-PK (1); The 14th road is Amino 650C-PK (2); The 15th road is Amino650C+PK (1); The 16th road is Amino 650C+PK (2)).Experimental result as shown in Figure 2 clearly illustrates that the existence of spacerarm is necessary for the PrPsc combination of Amino 650-M resin.

Embodiment 7

Catch PrPc existing under the human serum albumin of high concentration (HSA)

Resin is removed PrP from the treatment product that contains multiple proteins ability is described for example, has studied when having the human serum albumin, therefrom in conjunction with the effect of PrPc.

With four Bio-Rad ^TMPillar is used Toyopearl ^TMAmino 650M amino resins is filled.Resin height of bed 1cm, volume are 0.5ml.Resin fully washes with the work buffer.The sample that loads on the pillar is as follows:

1%nHaBH in the pillar I-working buffer liquid (normal hamster brain homogenate liquid);

Pillar II-1%HaBH is with the 25%HSA (Sigma) of work buffer preparation;

Pillar III-1%HaBH, and the 25%HSA (Sigma) of usefulness work buffer preparation and 20mM N-Ac-Trp (Acros Organics, Belgium);

Pillar IV-1%HaBH, American Red Cross preparation (ARC preparation).

20mM N-Ac-Trp is dissolved among the 25%HSA for preparing with the work buffer, vibration, 37 ℃ were heated 45 minutes.Prepare the 10%nHaBH supernatant as described above,, obtain 1% nHaBH with in the material that is diluted in selection at 1: 10 (step 2).

The bottom of each pillar links to each other with the peristaltic pump of 4 passages.5 milliliters of (5ml) 1%nHaBH of above step preparation pass through pillar I-IV with the flow velocity of 0.5ml/min.Pillar is washed, with 10ml working buffer liquid/pillar, with the washing of the flow velocity of 0.5ml/min.Reclaim resin, prepare sample as described above, run 12%Bis-Tris SDS-PAGE gel.

Use 3F4 one anti-Western trace to be used to detect the PrPc that has been caught by resin.The photo of trace is (the 1st road-low concentration Mus matched group as shown in Figure 3; The 2nd road-middle concentration Mus IgG matched group; The 3rd road-nHaBH matched group; The 4th road-1%HaBH pillar; The 5th road-1%HaBH, the 25%HSA pillar; The 6th road-1%HaBH, 25%HSA, 20mM N-AC-Trp pillar; The 7th road-1%HaBH, ARC preparation pillar).Can see the band of about equal densities in per pass, this shows: Toyopearl ^TM650-M amino resins under the condition that 25% human serum albumin who obtains from number of ways exists, has been caught the PrPc in the hamster brain homogenate liquid.

Experimental result as shown in Figure 3 shows: resin from the sample that contains HAS in conjunction with the ability of prion protein, thereby provide resin can be used at multiple treatment product in conjunction with prion protein, and guarantee to use the treatment product of haemproteins, for example as stabilizing agent or therapeutic agent, and the evidence that can be subjected to the treatment security of products that PrP pollutes.

Embodiment 8

In the human serum albumin, PrPsc is attached on the amino resins

In experiment described below, the combination of the infectiousness PrPsc in being incorporated in albumin has been described.With 12 PIKSI pillar Toyopearl ^TMAmino 650M resin is filled, each 0.5ml.2ml 10%SBH (sheep itching disease brain homogenate liquid) is handled with 0.5% sarcosyl.

According to following general introduction, prepare following six kinds of activators.

1. with the SBH activator of buffer preparation: supernatant of handling through sarcosyl with work buffer dilution (1: 100); 0.22ml SBH is added in the 22ml working buffer liquid.

2. use the SBH activator of HSA (American Red Cross (ARC) preparation) preparation: the supernatant that sarcosyl was handled dilutes (1: 100) with 25%HAS; 0.22mlSBH is added among the 22ml HSA (American Red Cross preparation).

3. use the SBH activator of HSA (Sigma) preparation that contains N-acetyl-DL-tryptophan and caprylate: the supernatant that sarcosyl was handled dilutes (1: 100) with the 25%HSA that contains 20mM N-acetyl-tryptophan and 20mM caprylate; Albumin is bought from sigma, does not contain additive; The SBH of 0.22ml is added in the HSA solution of 22ml.

4. use the SBH activator of HSA (Sigma) preparation that contains N-acetyl-tryptophan: the supernatant that sarcosyl was handled dilutes (1: 100) with 25%HSA; The SBH of 0.22ml is added to 22ml to be contained among the HSA of N-acetyl-tryptophan.

5. use the SBH activator of HSA (Sigma) preparation that contains caprylate: the supernatant that sarcosyl was handled dilutes (1: 100) with 25%HSA; The SBH of 0.22ml is added to 22ml to be contained in the HSA solution of 20mM caprylate.

6. with the SBH activator of independent HSA (Sigma) preparation: the supernatant that sarcosyl was handled dilutes (1: 100) with 25%HSA; 0.22ml SBH is added in the HSA solution (Sigma) of 22ml.

Every kind of resin 10ml solution repetition of activation 2 times.Pillar is with sample on the flow velocity of 0.5ml/min, with peristaltic pump control flow velocity.Collect and flow out solution, and collect from the resin of each pillar.Activator in every kind of resin and the buffer is imported protease K digesting liquid.According to method described here, sample carries out the Western trace.Trace is described (group A: the 1st road-molecular weight standard thing in Fig. 4; The SBH-PK that 0.1% sarcosyl of the 2nd road-buffer preparation is handled; The SBH+PK that 0.1% sarcosyl of the 3rd road-buffer preparation is handled; The SBH-PK (1) of the 4th road-buffer preparation; The SBH-PK (2) of the 5th road-buffer preparation; The SBH+PK (1) of the 6th road-buffer preparation; The SBH+PK (2) of the 7th road-buffer preparation; The SBH-PK (1) of the 8th road-HSA (ARC composition) preparation; The SBH-PK (2) of the 9th road-HSA (ARC composition) preparation; The SBH+PK (1) of the 10th road-HSA (ARC composition) preparation; The SBH+PK (2) of the 11st road-HSA (ARC composition) preparation; The SBH-PK (1) of the 12nd road-HSA (Sigma) preparation; The SBH-PK (2) of the 13rd road-HSA (Sigma) preparation; The SBH+PK (1) of the 14th road-HSA (Sigma) preparation; The SBH+PK (2) of the 15th road-HSA (Sigma) preparation; Row; Group B: the 1st road-molecular weight standard thing; The SBH-PK that 0.1% sarcosyl of the 2nd road-buffer preparation is handled; The SBH+PK that the 0.1%Sark of the 3rd road-buffer preparation handles; The SBH-PK (1) of HSA (Sigma) preparation of the 4th road-contain acetyltryptophan; The SBH-PK (2) of HSA (Sigma) preparation of the 5th road-contain acetyltryptophan; The SBH+PK (1) of HSA (Sigma) preparation of the 6th road-contain acetyltryptophan; The SBH+PK (2) of HSA (Sigma) preparation of the 7th road-contain acetyltryptophan; The SBH-PK (1) of HSA (Sigma) preparation of the 8th road-contain caprylate; The SBH-PK (2) of HSA (Sigma) preparation of the 9th road-contain caprylate; The SBH+PK (1) of HSA (Sigma) preparation of the 10th road-contain caprylate; The SBH+PK (2) of HSA (Sigma) preparation of the 11st road-contain caprylate; The SBH-PK (1) of HSA (Sigma) preparation of the 12nd road-contain acetyltryptophan and caprylate; The SBH-PK (2) of HSA (Sigma) preparation of the 13rd road-contain acetyltryptophan and caprylate; The SBH+PK (1) of HSA (Sigma) preparation of the 14th road-contain acetyltryptophan and caprylate; The SBH+PK (2) of HSA (Sigma) preparation of the 15th road-contain acetyltryptophan and caprylate.

Result shown in Fig. 4 shows that when mixing with the human serum albumin, infectiousness PrPres can be incorporated on the amino resins, and multiple additives can not disturbed combination.

Embodiment 9

Screening is used for combination and removes the resin of prion protein

Purpose

The level that the propagability spongiform encephalopathy infectiousness that this research is used for estimating the resin form selected by PRDT (Virus Removal And Diagnostic Technology Co., Ltd) to be provided is removed.Activation to this resin is the leukopenic human red blood cell concentrate unit that has mixed pruritus hamster brain homogenate liquid.

Background

Propagability spongiform encephalopathy (TSE) infectiousness is confirmed in the blood of the animal of several breadboard experimental and natural infections.Consistent and every milliliter of the infectiousness can measuring 4-20ID (infective dose) of BREF/UM by the hamster whole blood of 263K pruritus bacterial strain (263K strain ofscrapie) infection with reappearing.In addition, reported blood via vCJD (TSE of human form) from the people to anthrochorous two routine dubiety cases.These results have caused by blood and blood products TSE have been propagated problem to the mankind's potential risk.Because the TSE medicament is to the resistance of the pathogen inactivated method of tradition and lack sensitive incubation period of TSE diagnostic test, removing this medicament is to solve the most promising scheme that TSE propagates through blood.

The strategy of PRDT adopts and produce potential part from chemicals and peptide storehouse.Screening is used to have the part of PrPres in conjunction with character through firsts and seconds in this storehouse, by the elisa assay of the PrPres in proteic Western trace of resin-bonded and the effluent.PrPres is considered to the communicable biochemical marker of TSE.These residue on sieves have been differentiated and several TSE have been removed effective part.The purpose of this research is to determine to be used for removing the level that the TSE infectiousness that provides when using the communicable equipment of TSE is removed from leukopenic erythrocyte concentrate (RBCC) by the part of these selections.Select eight kinds of PRDT resins specificity from the brain solution of mixing people (sporadic CJD) among people RBCC and hamster (263K pruritus bacterial strain) and infecting to catch PrPres.The resin of these selections is: SYA, and Amino 650M, Phenyl 650M, TMAE 650, DVR, YVHEA, WFDEA and (D) ES (nal) PRQ-Eaca.Also comprise two kinds of negative control things: acetylation SYA and acetylation Amino 650M.Code Number is shown in Table 5.(using amino acid whose standard to write a Chinese character in simplified form) at this.Had been found that before that a large amount of acetylation acetic anhydrides suppressed prion combination.

Table 5 resin code

The resin code

SYA R1

Ac.SYA R2

Amino 650 R3

Ac.Amino 650 R4

TMAE R5

Phenyl 650M R6

DVR R7

YVHEA R8

WFDEA R9

(D)ES(nal)PRQ-Eaca R10

Experimental design

Resin compatibility-in preliminary study, at first test (current specifications) the RBCC function of each resin alternatives and current explanation and the compatibility of stability.All resins and RBCC compatibility also are used for TSE infectiousness removal research.In one embodiment, the PRDT resin is tested with two pillar series connection.Each pillar (or equipment) contains about 10mL resin.The ultimate principle of series connection pillar be used for type that determine to remove and difference at random or optionally catch mechanism.Suppose that resin is not under saturation conditions, in mechanism at random, second equipment is removed extra infectiousness.In selectivity mechanism, second equipment is not removed extra infectiousness.In one embodiment, two placed in-line TMAE resin columns come the comparative evaluation with a unitary RBCC who has mixed 1% pruritus brain homogenate liquid.Select this TMAE resin to be because its effectiveness.These resins show that second pillar has been removed with the as many PrPres of first pillar and through the liquid stream of second pillar and may contain more PrPres.According to these results, can reach a conclusion, first equipment is saturated.The PrPres removal level of each resin is determined by the Western trace, and is measured the resin that demonstration PrPres removal level is lower than Western trace detection level.

In one embodiment, whole experiment was finished in one day, and one group of blood is mixed and use with each test of opposing the activation of PRDT part at once.This strategy has effectively increased the value of this research with the Cost Growth of minimum.This research also is designed to activate the final application that each resin comes the actual capabilities of simulator with a unitary doping RBCC.

Select the filler of pruritus hamster brain homogenate liquid as RBCC.The ultimate principle of this decision based on: 1) pruritus hamster brain homogenate liquid has a high assay amount ideal value for mixing research, wherein filler must diluting several times, 2) the hamster model has the shortest culture period in the different experiments model of TSE bacterial strain, 3) part of Xuan Zeing from pruritus hamster brain homogenate liquid screening be used for the combination and 4 of PrPres) the hamster model established and a large amount of filler can be easy to preparation at laboratory.

The calculating of resin capacity (capacity)-theoretical resin capacity is carried out after infectiousness research.Usually, the resin that is used to study in theory can be in conjunction with the every gram resin of 30-40mg albumen (4.7mL swelling resin).In activator, select this capacity to come in conjunction with whole (PrPc+PrPres).Yet, calculate this capacity for albumen with ideal behavior (as separating) at resin hole of bead inside/outside.PrP is not ideal albumen in this activator the time, though with the set form of various yardsticks or with other proteic compound carry out clean after.Shown that also PrP can not penetrate the hole of bead and it is attached to the surface of resin bead.Although this limitation is arranged, find that 50mL resin (cumulative volumes of five equipment of combination) is enough in conjunction with all PrP in the activator.Filtering target flow velocity is 10mL/min.This flow velocity derives from the requirement at a unit blood of 40min inner filtration.Under these conditions, be about 1mL/min time of contact, and think that for infectiousness it is enough to binding partner.Western trace and the elisa assay of resin-bonded PrPres carry out in liquid stream.

Experimental arrangement

Blood converges-ten unitary people RBCC and is collected in ARC (Holland laboratory) and filters leukocyte according to standardization program well known in the art on the Pall filter.Blood in the sack is delivered to laboratory and store overnight in 40 ℃ refrigerator in experiment the previous day.Test the same day, blood is convergeed in the big blood bag.All dilutions and cubing are carried out with weight and are used 1.06g/mL to change into volume as the density of RBCC.

The doping prepared product, in the doping of blood and reallocation-unadulterated blood group (blood pool) equal portions take out and as concentration determination with the diluent in the serial dilution preparation.Equal portions in PRDT pruritus brain homogenate liquid (SBH) group were handled on ice 30 minutes with 0.5% sarcosyl.With this sample centrifugalize, shift out supernatant " 10%SBH that sarcosyl is handled " and also slowly mix with the blood group.The volume of filler calculates based on blood weight and makes that the final concentration in the blood is 0.1%w/v (1: 100 dilution of 10%SBH).After adulterated blood conscientiously mixed, blood converged bag (blood pool bag) and is connected to the manifold (manifold) that is beforehand with and adulterated blood is redistributed to ten blood bags, each about unit.Transmission is undertaken by gravity and the sack of filling is placed on the balance weighing.The blood of an about unit weight is sent in each sack, and final weight is recorded as Challenge 1.Take out the adulterated blood of equal portions and be used for concentration determination.

Each the blood bag that filters-contain doping RBCC is connected in the defecator that is beforehand with.Each defecator contains the same resin (the every post of～10mL resin) of placed in-line five pillars and five blood bags to collect the liquid stream from each filter process.Hang the blood bag, fixedly pillar is perpendicular to liquid stream with pliers, and the reception bag keeps flat from the teeth outwards.Unclamp two pliers that place the pillar above and below just and filter beginning.Timing is carried out in the filtration of each pillar, and the height between record sack and the pillar.Should be noted that pillar and the distance that receives between the bag are identical for all defecators.Under kindred circumstances, in filter process, the height that reduces or increase between blood bag and the pillar is regulated flow velocity.After each pillar filtered, weighing received bag, takes out an equal portions liquid stream, and the sack of weighing is once more also prepared for filtering next time.

After the removal of resin-each filtration, pillar is separated with other parts of device and place the next door to be used for removing subsequently resin.By slowly converging buffer in pillar and wait for that resin comes out, can from about 60mL buffer of each pillar, collect whole ten milliliters of resins.About 3-5mL resin is transferred to disposable chromatographic column and washs with citrate buffer.Abandon remaining resin.

Biochemical characteristic-by Western engram analysis resin catching for PrPres.The result shows SYA, Amino 650M, DVR, YVHEA and (D) ES (nal) PRQ-Eaca remove PrPres to detectability by second pillar.In other words, there is not the PrPres signal in the 3rd, the 4th and the 5th pillar, can detect.

In bench-scale testing, five effluent from four kinds of resin: DVR, phenyl resin, amino resins and TMAE are carried out elisa assay.The program of sample preparation is: add the water of 2 volumes, add 2%SDS (final concentration) and heat 10 minutes (TSE deactivation program) at 1000 ℃.Before being applied to ELISA, sample is diluted 50 times with analysis buffer.The final sample dilution is 150 times.These results and Western trace result's general trend matches.Best resin according to ELISA result is an amino resins, is DVR, TMAE and phenyl resin then.

Concentration determination

Concentration determination is with the selection of resin and liquid stream-based on the Western trace result of resin-bonded PrPres, find the 3rd pillar after five kinds of parts removed PrPres to detectability.These five kinds of parts are: DVR, Amino 650M, SYA, YVHEA and (D) ES (nal) PRQ-Eaca.The infectiousness of these five kinds of parts is removed with the TMAE and the acetylation SYA resin of thing carry out in contrast.For reducing the total quantity of animal in the research, be not that per share effluent all carries out concentration determination.Based on Western trace result, abandon liquid stream #1, because being all PrPres and possible infectiousness, do not removed the strong PrPres signal indicating in the post 2 by post 1.The test effluent of selecting is 2 and 5.Select effluent 2 to be because (except that tester) post 3 shows signal hardly in all cases shows that PrPres concentration has obvious reduction in the liquid stream 2.Selecting liquid stream 5 is because it has the best infectious chance of removal.In research afterwards, effluent 3 and 4 also carries out concentration determination.Yet this report only relates to infectiousness research for the first time.

The concentration determination of the concentration determination-effluent by the culture period method utilizes the culture period method to carry out.This method is not enough to very distinguish accurately two close concentration determination amount (2-log ₁₀Or lower), but it can ideally be used for difference and is 3-log at least ₁₀Communicable concentration determination is because it compares requirement animal still less fast and with terminal method for measurement of concentration more accurately.Because the purpose of this research is to select to remove greater than 3-log ₁₀Communicable resin thinks that culture period is correct selection.Culture period is inversely proportional to the infectiousness dosage that is inoculated into hamster.This relation is linear for highly infective dosage and should linearity was what to separate with low infectiousness dosage.Adulterated RBCC be diluted to two of the RBCC that do not mix independently ten times of dilution series (with respect to whole brain from 10 ^-3To 10 ^-11) carry out concentration determination and produce two dose-effect curves according to culture period.

Animal is separated into to be equal to have determined to improve cultivates the statistical validity that time value and terminal diluted concentration are measured.The result of calibration trace has determined for each dilution from activator (10 ^-3) begin and 10 as maximum concentration ^-4, 10 ^-5Deng the average culture period of disease.Use two identical serial dilutions to determine the accurate concentration determination of filler by terminal concentration determination method.Final removal utilization through resin is determined from the dose-effect curve equation of the culture period experiment acquisition of serial dilution challenge.

It is terminal that concentration determination-in identical experiment, 10% pruritus brain homogenate liquid and sarcosyl supernatant carry out concentration determination with terminal concentration determination method.The ultimate principle of the concentration determination of these increases is the accurate measurements for the initial concentration mensuration that obtains PRDT SBH pool and the concentration determination of the actual filler that does not contain RBCC.

Inoculation-effluent is inoculated without dilution.Two cage animals (8 hamsters) inoculate with effluent, and 4 cage animals (16 hamsters) inoculate with challenge solution.The liquid stream 2 that contains amino resins is inoculated in 4 cages (16 hamsters) lining.Preparation two of dose-effect curve is dilution series independently.Two cage animals are used from 10 ^-3Begin to 10 ^-11Each diluent (diluent with whole brain as 10 ⁰Dilution) inoculation.For the concentration determination of SBH and sarcosyl SBH, 1 cage animal is with 10 ^-1To 10 ^-11Diluent inoculation.

Weight data-previous work shows that the body weight of suffering from the pruritus animal is along with advancing of disease descends.Decision uses this standard to determine the end of cultivating.The weight of animals drops to 80% the end that is defined as culture period that day of weight limit.The advantage of this method is that it does not have bias and not influenced by human error.Each animal is weighed weekly, and once there being disease indication to weigh twice weekly, the record body weight is also mapped based on days post inoculation for each animal.Incorporate last hurdle into each animal of effluent inoculation and the final culture period of the animal in the standard curve, title is " 80% maximum ".The days post inoculation of record is on " PI natural law " hurdle, and " 80% maximum " be not always corresponding, because some animals do not die when body weight drops to 80% weight limit, but after several days.Yet, before reaching target weight, do not have animal to die.Days post inoculation in " 80% maximum " hurdle is according to showing that first day the body weight table that animal reaches target weight determines.The average of these numerical value and standard deviation are also measured and are listed in the following table 6.

Clinical data-with the animal of effluent inoculation is in inoculation death in back 239 days.Terminal concentration determination is proceeded, and will determine the accurate concentration determination of SBH over continuous 365 days.Yet these remaining animals are no longer weighed.There are not concurrent death and 7 animal the last fortnight death after inoculation owing to hematotoxicity.These animals are measured the calculating from final concentration and remove.

With or the concentration determination of the SBH that handles without sarcosyl undertaken by clinical score biweekly animal, can not stand up to animal, this moment, animal die.This animal semiography is scanned and submits to.

The culture period of each diluent of table 6 in dose-effect curve

The dilution log of SBH among the RBCC 10	(I)		(II)
					Meansigma methods	SD	Meansigma methods	SD
	-3 -4 -5 -6 -7	94 105 114 128 137	4.2 8 5.2 9.4 15	99 104 111 117 158					5.4 6 3.8 4.8 54

-8 -9 -10

227 147 151

49 - -

182 206.5 0

73 104 -

Calculate and the result

The calculating of terminal concentration determination-SBH concentration determination utilizes Reed and Muench method to carry out with the 1g brain.Four groups of data have been produced in the concentration determination method endways: the dilution of two standard curves, a 10%SBH and a dilution through the 10%SBH supernatant of sarcosyl processing.The latter is the actual filler among the RBCC.Table 7 has shown the tabulating result of concentration determination.The concentration determination amount of PRDT 10%SBH pool is 10 ^9.18ID/mL.After sarcosyl was handled and removed insoluble particles, the concentration determination amount reduced to 10 ^8.10ID/mL is although show the infectiousness (90%) that has solubilising program centrifuging to remove about 1-log.The concentration determination of Challenge solution (0.1%SBH) is carried out twice demonstration and is well mated, and twice concentration determination amount all is 10 ^6.72ID/mL.This concentration determination amount is slightly higher than in buffer the calculating concentration measured quantity of the SBH that 100 times of dilutions to 0.1% sarcosyl handles.Not clear what reason causes this difference.

The concentration determination amount of the terminal dilution method of table 7

	The concentration determination amount, log 10
		The 0.1%SBH (II) of the 0.1%SBH (I) of 10%SBH mother solution 10%SBH sarcosyl supernatant in RBCC in RBCC	9.18 8.10 6.72 6.72

Culture period concentration determination-in the culture period concentration determination, use the log of dilution ₁₀Dose-effect curve utilizes activator from 10 ^-3To 10 ^-8The experiment culture period of each diluent determine.The meansigma methods of two curves of table 6 record is averaged (table 8).

Two average culture periods of curve of table 8

-3 -4 -5 -6 -7 -8

96.5 104.5 112.5 122.5 148.5 204.5

Extract the curve of numerical value in the most suitable table 8 and the exponential curve of its corresponding following equation out:

y＝a+b ^(-x/c)

Wherein, a, b and c are the constants with report numerical value, and y relates to the dilution of whole brain, and (that is, 0.1%SBH is 10 ^-3The dilution of whole brain) and x be culture period.The meansigma methods and the standard deviation of liquid stream culture period are shown in table 9.Dilution corresponding to each culture period utilizes above-mentioned equation to calculate.The difference of the dilution of per share liquid stream and activator dilution is calculated as removal.

Remove result's (table 9) and show that liquid stream 2 catches one or less than 1-log ₁₀Infectiousness, wherein R3FT2 (Amino 650M resin) is best.Also have the negative control resin, R4FT2 (acetylation amino resins) does not remove infectiousness and R5FT2 (TMAE) only removes the infectiousness of 0.5-log.On the other hand, liquid stream 5 shows R1, R3, and R7 and R8 (being respectively SYA, Amino 650M, DVR and YVHEA) all remove greater than 3-log ₁₀Infectiousness, wherein SYA is best, is 4.2-log ₁₀Remove.

Table 9TSE infectiousness is removed

	Resin	DPI *	SD	Dilution (log 10)	Remove (log 10)
						R1FT2 R3FT2 R4FT2 R5FT2	Activator SYA Amino 650M acetylation Amino 650M TMAE	89 102 104 92 95	2.6 7.9 4.2 3.6 6.7	3 3.7 4.0 3.0 3.0	- 0.7 1.0 0.0 0.0

R7FT2 R8FT2 R10FT2	DVR WFDEA (D)ES(nal)PRQ-Eaca	102 100 98	2.7 5.0 1.4	3.7 3.6 3.3	0.7 0.6 0.3
						R1FT5 R3FT5 R4FT5 R5FT5 R7FT5 R8FT5 R10FT5	SYA Amino 650M acetylation Amino 650M TMAE DVR WFDEA; (D) ES; (nal) PRQ-Eaca	151 142 95 99 130 139 123	34.1 35.3 3.6 3.5 7.7 27.8 11.5	7.2 6.9 3.0 3.4 6.4 6.8 5.8	4.2 3.9 0.0 0.4 3.4 3.8 2.8

^*The DPI=days post inoculation

Conclusion

These central best PRDT parts that are selected from the secondary screening that is used for effective TSE removal have been differentiated in this research.When catching and removing of PrPres determined in the secondary screening, the infectiousness of identical ligands removal doping RBCC had also been confirmed in this research.These resins have been realized the purpose of this research, because most of part is removed greater than 3-log ₁₀The infectiousness from brain, and Amino650M and SYA remove about 4-log ₁₀In addition, this removal is specific, and non-by the big aggregation of size exclusion, because negative control thing part is not removed any infectiousness.Data show that also part does not have to remove the whole infectiousness that are present in the activator.This no wonder is because resin is by the very infectiousness activation of high concentration.The concentration determination amount of the activator of measuring based on Reed and Muench method is exposed to 10 with resin ^6.72ID/mL, it is 100,000 times of the infectiousness level (10ID/mL) measured in the blood of the rodent model that TSE infects.

In a word, SYA, Amino 650M, DVR and YVHEA all do well, wherein SYA and Amino 650M preference slightly.These resins are removed 3-4-log ₁₀The TSE infectiousness.This removal level is enough to catch whole infectiousness of autoblood, because a unitary infection blood (in the hamster model) contains about 5,000 infective doses.Thereby if the infectiousness in the brain is similar to the infectiousness in the blood, expection PRDT equipment is removed whole TSE infectiousness from blood.

Embodiment 10

From mixing buffer, filtering PrPsc and Amino 650M and the bonded comparison of Amino 650U among the SBH in blood plasma and the whole blood

Amino 650U is the mixture that comprises the different average bead size of Amino 650M, and its production cost is lower than 650M.The endogenous PrP of test Amino 650U and in conjunction with the ability of the PrPsc in the substrate of all current uses (buffer, filter blood plasma and whole blood), and with itself and with compare with the combination of the activatory Amino 650M of doping whole blood.Design this experiment and be used to contrast, and establish from the endogenous PrPc of blood plasma and whole blood and combining of Amino 650U from the PrPsc of adulterated buffer, blood plasma and whole blood and combining of Amino 650U.In addition, design the effect that this tests to determine leukoreduction filter (leukofiltration) in the PrPc removal.

In the time of in being present in blood plasma or whole blood, do not find that the signal that the Protein virus of 650U or 650M removes is variant.In a word, Amino 650U is identical with the 650M performance.The amount of the PrPc that leukoreduction filter is removed is greater than the estimator of platelet and leukocyte one time-out.Thereby leukoreduction filter also may be caught the PrPc in part blood plasma source.Show that the model of action of leucocyte filter is different when catching the PrPc in people and hamster blood plasma source.When hamster blood plasma PrPc was not filtered device and catches, human plasma PrPc may be hunted down.At last, also possible, two results' difference is owing to lack dependency between PrPc and the infectiousness.

Embodiment 11 hamster brain PrPsc combine with the AMN resin

To a series of resins (as, AMN-13,14,15,16 and 17, Amino 650M and Amino 650U) carry out comparable in conjunction with experiment.The PrPsc of buffer, blood plasma and the whole blood of these resin-bonded arrival auto-dopes.The result has confirmed that when activating simultaneously with adulterated buffer and adulterated whole blood all the AMN resin-bonded is equally good.In addition, the signal of AMN resin is identical with the signal of Amino 650M and 650U.Contrast finds that from the resin-bonded of the PrP in the adulterated blood plasma Amino 650M is slightly stronger than the signal of every other resin.In the AMN resin, #13 shows to have weak PrP signal, but very suitable with Amino 650U, and #15,16,17 all outperform Amino 650U.At AMN 14,15,16,17 interlaminar resins do not discover a marked discrepancy.

In a word, this studies confirm that more similaritys of interlaminar resin, and very important, and it has shown with the dependency of Amino 650U more approaching than 650M.Observed and difference blood plasma show that at least perhaps the difference with the activator that reduces substitution level be favourable, and the resin performance more approach Amino 650M.

Embodiment 12 is bonded to the proteic extraction of the film that resin implants and determines from the PrPc of normal hamster brain homogenate liquid is bonded

The demand for development exploitation of the new equipment of the calendered film that the use resin is implanted is extracted protein-bonded new method from resin.Must change processing to material, and the composition of extracting solution, concentration and volume.Experiment also is designed to be used for carry out combination evaluation with new model, the film and the acetylated form thereof that use Toyopearl Amino650M to implant.

Normal hamster brain homogenate liquid (HaBH) is handled and rotation with sarcosyl.It is 1% that the supernatant that forms is diluted to final concentration with work buffer or people's whole blood.50 milliliters of adulterated flow of solution are through 47mm Swinnex filter supporter (Millipore), and it contains 4 has implanted 4mg/cm ²Toyopearl Amino 650M or the sandwich of the calendered film of its acetylated form.The flow velocity that uses is 0.5mL/min, uses peristaltic pump.Collect ten equal portions, each 5mL for each adulterated solution and film type.Liquid stream sample elisa assay with the activatory film of adulterated buffer.Contain the acetylation resin and use working buffer liquid to clean with the activatory film of adulterated whole blood.

Film part (being monoblock in some cases) is handled with SDS-PAGE sample buffer or 99% formic acid.Comprise with the processing of formic acid the formic acid of 0.5mL 99% and 10 μ L 20%SDS being joined 1/4th film sandwich, then cultivate and removed liquid in 1 hour, use the SpeedVac evaporation.Make water with the volume-adjustment of sample to 15 μ L, then add 15 μ L 2X sample buffers.Use the processing of sample buffer to comprise 3mL 1X sample buffer is joined in the monoblock film, then cultivate 30 minutes, and seethed with excitement 7 minutes.Squeeze film is not collected solution, simple centrifugal all resins of removing.Also tested the variation of above-mentioned processing.It comprises 1mL 2X sample buffer is joined in two isolating films corresponding to the 1A filter, cultivates 1 hour, in them one of then only seething with excitement.If split the filter supporter danger close that becomes when utilizing infectiousness, do not seethe with excitement and to use with the elution of sample buffer.

The final condition of test is to examine and the combining with contact activation solution of the first, second, third and the 4th film with the part (1/4) of sample buffer culture membrane.Then sample is moved on the SDS-PAGE gel and to whole protein staining.Also carry out the Western trace.The idle volume of filter supporter approximately is 7mL.The 50mL activated solution is flowed through behind each filter, is that air flows through then, and the activated solution of recovery is 45 and 47mL for whole blood.When using adulterated buffer, the volume of recovery is 46 and 46mL.When using different activated solution, do not notice evident difference.

The first filter supporter of opening is that of film that contains band acetylation Toyopearl, and it activates with adulterated whole blood.Notice,, still have some blood in the filter although flow through air and use buffer solution for cleaning.In the process of attempting with the buffer solution for cleaning film, resin has significantly sacrificing, abandons film.

The filter supporter that has with whole blood activated T oyopearl Amino 650M uses extra 200mL buffer solution for cleaning.Flow velocity is higher than maximum (graduated disc is 999).When opening supporter, notice particularly still having some blood in each layer between each layer.Be also noted that and in washing process, walked around a pair of part of delineating by the ray allotter.

The film piece is cut into 4 equal portions.A slice have isolating and handle with sample buffer four layers to determine whether different layers has different combinations.Another equal portions also are divided into small pieces and handle with formic acid.The processing that remaining two equal portions are used for relatively heating and do not heat.

Two each use 200mL working buffer liquid of the activatory filter of the adulterated buffer of usefulness clean.Open filter and monoblock is transferred in the vial, add the 3mL sample buffer therein.

The resin that is implanted to calendered film shows keeps identical PrP in conjunction with character in the resin of cylindricality formula.Acetylation amino resins shows that the weak film suitable with the amino resins signal combines the PrP signal, confirms as drawing a conclusion: acetylation amino resins can be not effectively have specificity in conjunction with the difference between PrP and two kinds of signals to amino resins.Usually, resin shows 50% acetylation, no matter is mixed form or chemosynthesis, reduces the PrPres combination.

Embodiment 13PrPsc and combining from the D4 of adulterated blood plasma of SBH and whole blood

Experimentize to come relatively from combining of the PrPres of adulterated buffer, filtering blood plasma and whole blood and D4 resin and combining of endogenous PrPc and D4.In the Prometic simulation resin with adulterated RBCC (UM-R-T-SE-180603) test, D4 demonstrates the best incorporated with PrPsc.This resin is carried out the research of endogenous infectiousness.Test in the presence of blood plasma, whole blood with the D4 binding ability of adulterated brain source PrPres, buffer is thing in contrast.This test also only uses the endogenous PrPc from blood plasma or whole blood to activate D4.

Western trace signal indicating D4 is in conjunction with the doped P rPres in blood plasma and the whole blood.D4 also may may be better than Amino 650M in conjunction with the endogenous PrPc from whole blood.

Embodiment 14 different acetylation resins catching to PrPres

This research is compared the acetylation of Amino 650M to being bonded to the influence of PrPres.The following realization of acetylation in various degree (1) fully mixture and (2) of acetylation and non-acetylation resin by on identical pearl, carrying out the part chemical reaction.This result shows that resin can stand 20% acetylation that is realized by chemical reaction, still in conjunction with PrP.In addition, the mixture of 50% acetylation resin is for also being gratifying in conjunction with PrP.

Though can use method and the material similar or suitable with material to method described here in practice of the present invention or the check, described above is suitable method and material.The reference material of all publications, patent application, patent and other citation referred in this intactly is incorporated in this, as a reference.In addition, material, method and embodiment only are illustrative, and its purpose does not lie in restriction.

Above-mentioned description is used to describe and invents relevant several embodiments.Can carry out many places to embodiment and structure and revise, add and deletion, only otherwise break away from scope of invention and spirit.

Sequence table

＜110〉American Red Cross

North Carolina State University

Prometic Biosciences Ltd.

＜120〉prion protein binding materials and using method thereof

<130>51821-0111WP(51821-299536)

<150>US 60/460,474

<151>2003-04-04

<160>1

<170>PatentIn version 3.2

<210>1

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic

<400>1

Pro His Gly Gly Gly Trp Gly Gln

1 5

Claims (20)

1. one kind is detected and the method for separating prion protein from sample, described method is included in and makes between prion protein and the polymerization prion protein binding materials under the bonded condition, contact described sample with this polymerization prion protein binding materials, wherein this polymerization prion protein binding materials comprises the substrate that is bonded to functional group, and wherein said functional group comprises hydrophilic group, hydrophobic group or amphiphile function group.

2. the method for claim 1, wherein this functional group is positively charged group, electronegative group or charged group not.

3. the method for claim 1, wherein this functional group is amido, sulphite group, sulfonyl, 2,2,2-HFC-143a sulfonyl, alkyl, aromatic group, siloxy group or fluorinated groups.

4. method as claimed in claim 3, wherein said aromatic group is a phenyl.

5. method as claimed in claim 3, wherein said alkyl is a butyl.

6. the method for claim 1, wherein said functional group is an amido.

7. method as claimed in claim 6, wherein amido is primary amine group, secondary amine group, tertiary amine group or quaternary amines.

8. method as claimed in claim 7, wherein quaternary amines is diethylamino ethyl, dimethyl aminoethyl or trimethyl amino-ethyl.

9. the method for claim 1, wherein said functional group comprises:

a)-OCH ₂-CHOH-CH ₂NH ₂；

b)-C ₆H ₅；

c)-(CH ₂) ₃-CH ₃；

d)-CH ₂-CH ₂-N ⁺H(C ₂H ₅) ₂；

e)-SO ₂-CH ₂-CF ₃；

f)-CH ₂-CH ₂-N ⁺H(CH ₃) ₂；

G)-CH ₂-CH ₂-N ⁺(CH ₃) ₃Or

h)-SO ₃ ^2-。

10. the method for claim 1, wherein said substrate is polymethylacrylic acid or methacrylic acid.

11. the method for claim 1, wherein said substrate is FRACTOGEL ^TMEMD, TOYOPEARL ^TMPerhaps TSK-GEL ^TMPolymeric matrix.

12. method as claimed in claim 11, wherein said polymeric matrix is TOYOPEARL ^TMAMINO 650.

13. the method for claim 1, wherein said prion protein are PrPc, PrPsc, PrPr or PrPres.

14. the method for claim 1, wherein said polymerization prion protein binding materials are arranged on chromatographic column, the film, on the fiber, on the pearl, inject non-woven screen cloth, coated fiber is included among the filter housings, or its combination.

15. the method for claim 1, wherein said sample are biological sample, food, environmental sample or water sample.

16. method as claimed in claim 15, wherein said biological sample derives from the human or animal.

17. method as claimed in claim 16, wherein said animal are cattle, sheep, pig, horse, Mus or animal in deer family.

18. the method for claim 1, wherein said prion protein are people, cattle, sheep, pig, horse, Mus or animal in deer family prion protein.

19. method as claimed in claim 18, wherein said biological sample are the sample that derives from blood, the sample that derives from brain, humoral sample, collagen extract, body of gland extract, tissue homogenate thing or extract.

20. method as claimed in claim 19, wherein said body fluid are blood, blood plasma, serum, cerebrospinal fluid, urine, saliva, milk, pipe liquid, tear or seminal fluid.

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